Use of an inhibitor of METTL1 in the preparation of a medicament for treating inflammation after central nervous system injury

Abstract

The present invention relates to the use of an inhibitor of METTL1 in the preparation of a medicament for treating inflammation after central nervous system injury, belonging to the technical field of biomedicine. The present invention has confirmed that central nervous system injury can lead to an increase in the expression level of METTL1; compared with the mice in the sham operation group, the 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. The present invention has found that METTL1 plays a key role in the inflammation of central nervous system injury, and reduces the secondary pathological changes of central nervous system injury by regulating the level of m 7 6G methylation, and this discovery provides a new target for the treatment of central nervous system injury, with important clinical application potential and broad market prospects.

Description

Technical Field

[0001] The present invention relates to the use of an inhibitor of METTL1 in the preparation of a drug for treating inflammation after central nervous system injury, and belongs to the field of biomedical technology. Background Art

[0002] Central nervous system (CNS) injury refers to the injury of the brain and spinal cord, mainly 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.). It is the main cause of death and disability globally. Although many current clinical treatments have alleviated the loss of nerve function to a certain extent, the long-term prognosis of CNS injury and the recovery of nerve 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 directly caused by external forces (such as car accidents, falls, etc.), including the cutting, tearing, or stretching of axons. Secondary injury is the secondary injury cascade reaction triggered on the basis of primary injury, and these reactions include inflammation, oxidative stress, apoptosis, and the activation of glial cells. More research on CNS injury inflammation and regeneration includes spinal cord injury, traumatic brain injury, etc. Among them, spinal cord injury (SCI) refers to severe nerve function disorders caused by damage to the spinal cord due to various factors, resulting in the destruction of nerve conduction tracts, and is characterized by high incidence and high disability; traumatic brain injury (TBI) is the damage to the brain structure and function caused by external forces (such as car accidents, falls, impacts, etc.), and its pathological features include brain tissue contusion, intracranial hematoma, and increased intracranial pressure. The overwhelming inflammatory response in the early stage of CNS injury, accompanied by the extensive upregulation of cytokines / chemokines and immune cell infiltration, will lead to further damage to neurons and surrounding tissues. Many studies have also shown that inflammation is a huge obstacle to axon regeneration and affects 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] In inflammation, the classical NF-κB pathway mainly stimulates pro-inflammatory receptors (such as TNF receptor superfamily, Toll-like receptor family (TLR)) and cytokine receptors of interleukin, recruits adapter proteins to the cytoplasmic domain of the receptor, and in turn recruits the IKK complex. The IKKβ subunit in the IKK complex phosphorylates IκB (mainly IκBα), leading to its ubiquitination and then degradation by the proteasome. The degradation of IκB releases the NF-κB dimer (p65 / p50), enabling it to translocate from the cytoplasm to the nucleus. In the nucleus, the p65 / p50 dimer binds to the promoter of genes with κB sites, regulating the transcription of these genes and thus participating 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 the key subunit that enters the nucleus and regulates gene transcription. At the same time, the phosphorylation modification of p65 has an important impact on its function and activity, which can enhance its binding ability to DNA and transcriptional regulatory activity. Therefore, the changes in the expression levels of p65 and p-p65 directly affect the NF-κB pathway. By regulating the NF-κB pathway, the damage of inflammation to the injured tissue can be reduced, and the prognosis and nerve function recovery can be improved.

[0005] In recent years, the regulatory role of epigenetic modifications in the occurrence and development of diseases has attracted much attention. N 7 -methylguanosine (m 7 G) is one of the most common RNA modifications and has recently attracted extensive attention. More and more evidence indicates 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. m 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, etc.). Among them, the m 7 G modification of mRNA is dynamically regulated by methyltransferases, and the most obvious one is methyltransferase-like enzyme 1 (METTL1). METTL1 forms a functional methyltransferase complex with cofactor WD repeat domain 4 (WDR4) to catalyze m 7N7-methylguanosine methylation affects the production, processing, and maturation of mRNA, and ultimately mediates multiple key biological processes. Recent evidence has shown that increased METTL1 expression is associated with the occurrence of several types of cancers. For example, in glioma research, it was found that METTL1 is highly expressed in glioma patients and increases with the increase of glioma grading. At the same time, there is an obvious correlation between the expression level of METTL1 and the clinical risk factors and prognosis of glioma patients. The expression of METTL1 may be an independent risk factor for the prognosis of glioma patients. (Zhang Miao, Wu Rui, Liu Yue, et al. Research progress on the epigenetic modification of N7-methylguanine 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, the role of METTL1 and its mediated m 7 G modification in neurological inflammation or acute and chronic inflammation remains to be explored.

[0006] Currently, there are no reports on METTL1 and its related effects on the inflammatory or NF-κB pathway, and there is also no relevant 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 therapeutic targets and experimental bases 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 an inhibitor of METTL1 in the preparation of a drug for treating inflammation after central nervous system injury, providing a new molecular target for the treatment of central nervous system injury.

[0008] The technical solution of the present invention is as follows:

[0009] Use of an inhibitor of METTL1 in the preparation of a drug for treating inflammation after central nervous system injury.

[0010] Preferably according to the present invention, the inhibitor of METTL1 can inhibit the NF-κB pathway of the inflammatory pathway by inhibiting the expression of METTL1, inhibit the phosphorylation of p65, and reduce the degree of inflammation after central nervous system injury.

[0011] Preferably according to the present invention, the inhibitor of METTL1 reduces the m 7 G methylation level by inhibiting the expression of METTL1 and plays an anti-inflammatory role.

[0012] Preferably according to the present invention, the inhibitor of METTL1 includes siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

[0013] More preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3.

[0014] More preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0015] More 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 as shown in SEQ ID NO.1; shMETTL1 is shRNA interfering with the expression of METTL1.

[0016] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases, etc.

[0017] Use of an inhibitor of METTL1 in the preparation of a drug for promoting nerve regeneration after central nervous system injury.

[0018] Preferably according to the present invention, the inhibitor of METTL1 can promote the expression of neurotrophic factors by inhibiting the expression of METTL1, thereby promoting nerve regeneration.

[0019] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases, etc.

[0020] 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.

[0021] Preferably according to the present invention, the inhibitor of METTL1 includes siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

[0022] More preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3.

[0023] More preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0024] 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 as shown in SEQ ID NO.1; shMETTL1 is an shRNA that interferes with the expression of METTL1.

[0025] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases, etc.

[0026] Beneficial effects:

[0027] 1. Taking spinal cord injury and traumatic brain injury as examples, the present invention proves that central nervous system injury can lead to an increase in the expression level of METTL1; further studies the therapeutic effect of METTL1 inhibitors on central nervous system injury. Compared with the mice in the sham operation group, the 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. Mechanistically, the knockdown of METTL1 leads to a decrease in m 7 6G methylation level, and can inhibit the phosphorylation of p65, inhibit 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, inflammatory factors, the NF-κB inflammatory pathway and central nervous system injury. The inventor's research on METTL1 and central nervous system injury models is pioneering.

[0028] 2. The present invention constructs an adeno-associated virus vector with specific knockdown of METTL1 in astrocytes. This 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 axonal regeneration, and promote the recovery of motor function, and has good application prospects.

[0029] 3. The present invention discovers that METTL1 plays a key role in the inflammation of central nervous system injury, and reduces the secondary pathological changes of central nervous system injury by regulating the level of m 7 6G methylation. This discovery provides a new target for the treatment of central nervous system injury, and has important clinical application potential and broad market prospects. Description of the drawings

[0030] Figure 1 It is a bar chart of the mRNA expression level of METTL1 in spinal cord tissue.

[0031] Figure 2Western Blot detection graph of METTL1 protein in spinal cord tissue.

[0032] Figure 3 Bar graph of mRNA expression level of METTL1 in brain tissue.

[0033] Figure 4 Bar graph of mRNA expression level of inflammatory index IL-6 after knocking down METTL1 in spinal cord tissue.

[0034] Figure 5 Bar graph of mRNA expression level of inflammatory index IL-1β after knocking down METTL1 in spinal cord tissue.

[0035] Figure 6 Bar graph of mRNA expression level of inflammatory index CCL2 after knocking down METTL1 in spinal cord tissue.

[0036] Figure 7 Bar graph of mRNA expression level of inflammatory index CXCL2 after knocking down METTL1 in spinal cord tissue.

[0037] Figure 8 ELISA detection graph of IL-1β after knocking down METTL1 in spinal cord tissue.

[0038] Figure 9 ELISA detection graph of IL-6 after knocking down METTL1 in spinal cord tissue.

[0039] Figure 10 ELISA detection graph of CCL2 after knocking down METTL1 in spinal cord tissue.

[0040] Figure 11 Bar graph of mRNA expression level of axon regeneration index NGF after knocking down METTL1 in spinal cord tissue.

[0041] Figure 12 Bar graph of mRNA expression level of axon regeneration index BDNF after knocking down METTL1 in spinal cord tissue.

[0042] Figure 13 BMS evaluation score curve of mice after knocking down METTL1 in spinal cord tissue.

[0043] Figure 14 ELISA detection graph of IL-1β after knocking down METTL1 in brain tissue.

[0044] Figure 15 ELISA detection graph of IL-6 after knocking down METTL1 in brain tissue.

[0045] Figure 16 Bar graph of the mRNA expression level of NGF, an axon regeneration index, after knocking down METTL1 in brain tissue.

[0046] Figure 17 Bar graph of the mRNA expression level of BDNF, an axon regeneration index, after knocking down METTL1 in brain tissue.

[0047] Figure 18 Bar graph of the mRNA expression level of IL-1β, an inflammatory index, after knocking down METTL1 in primary astrocytes.

[0048] Figure 19 Bar graph of the mRNA expression level of CXCL2, an inflammatory index, after knocking down METTL1 in primary astrocytes.

[0049] Figure 20 Bar graph of the mRNA expression level of IL-6, an inflammatory index, after knocking down METTL1 in primary astrocytes.

[0050] Figure 21 Bar graph of the mRNA expression level of CCL2, an inflammatory index, after knocking down METTL1 in primary astrocytes.

[0051] Figure 22 Western Blot detection graph of p-p65 and p65 proteins after knocking down METTL1 in primary astrocytes and stimulating with TNF-α.

[0052] Figure 23 For primary astrocytes after knocking down METTL1, m 7 Dot Blot detection graph of G methylation.

[0053] Figure 24 Bar graph of the mRNA expression level of METTL1 after stimulating primary astrocytes with TNF-α at different time gradients.

[0054] Figure 25 For primary astrocytes after stimulating with TNF-α at different time gradients, m 7 Dot Blot detection graph of G methylation.

[0055] Figure 26 For m 7 Dot Blot detection graph of G methylation in spinal cord tissue.

[0056] Significance analysis in the above figures: P>0.05 is ns, P<0.05 is *, P<0.01 is **, P<0.001 is ***, P<0.0001 is ****. Detailed implementation methods

[0057] The technical solution of the present invention will be further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto. Reagents and consumables involved in the embodiments are all ordinary commercially available products unless otherwise specified; steps and experimental operations involved in the embodiments are all conventional technical operations in the art unless otherwise specified.

[0058] This embodiment provides a target that can significantly inhibit the inflammatory process after central nervous system injury and improve nerve function recovery, and specifically, it is illustrated by taking the spinal cord injury (SCI) and traumatic brain injury (TBI) models of C57BL / 6J mice as representatives.

[0059] Example 1: The expression of METTL1 in astrocytes increases after central nervous system injury

[0060] 1.1. Construction of spinal cord injury model

[0061] First, clean the operating table and sterilize the required surgical instruments in advance by high-pressure steam sterilization. Prepare a sterile surgical drape, suture, suture needle, and iodophor. Fast the mice 8 hours before the operation to avoid vomiting or aspiration during the operation. Prepare a 3% pentobarbital solution with distilled water and inject it intraperitoneally into the mice at a dose standard of 30 mg / kg for anesthesia. Clamping the four limbs without response indicates complete anesthesia. Shave the hair on the back of the mice to expose the surgical area. Fix the mice in the prone position on the operating table to ensure the stability of the mice's position during the operation. Disinfect the surgical area with iodophor cotton balls to reduce the risk of contamination during the operation. Lay a disposable sterile surgical drape and make a 1.5 cm incision at the T9 segment of the spine along the midline of the back, bluntly separate the muscles and fascia layer by layer until the spinous process is exposed. Use a fiber forceps to perform a T9 laminectomy under a stereomicroscope to expose the spinal cord. Mice in the Sham group were not subjected to spinal cord injury after laminectomy, clean the injured area with sterile saline, suture the surgical incision layer by layer with sterile suture, and disinfect with iodophor cotton balls. Mice in the SCI group were struck on the spinal cord using a pneumatic spinal cord injury striker (68100, RWD, Shenzhen, China) at a striking speed of 1 m / s, a striking depth of 2 mm, and a dwelling time of the striker of 1 s. The cleaning, suturing, and disinfection steps and operations were the same as those in the Sham group. Place the mice on a heating blanket for 2 - 3 hours, and place them in a breeding cage after they wake up (mice can freely obtain food and water in the cage). To prevent urinary tract infection in mice, manually squeeze the bladder of the mice twice a day to help them urinate until the mice are sacrificed.

[0062] 1.2 Perform pre-experiment verification in the spinal cord injury model

[0063] Mice in the Sham group and the SCI group were sacrificed by intraperitoneal injection of 3% pentobarbital solution on the 3rd and 7th days after spinal cord injury. The spinal cord tissue at the injury site was taken for qPCR and Western Blot to detect the expression levels of METTL1 mRNA and protein.

[0064] The qPCR results showed that, Figure 1 compared with the sham operation group (Sham group), the mRNA level of METTL1 in mice of the SCI group gradually increased on the 3rd and 7th days after spinal cord injury.

[0065] The Western Blot detection results showed that, Figure 2 it was confirmed that the protein expression level of METTL1 increased on the 3rd and 7th days after spinal cord injury.

[0066] 1.3 Construction of traumatic brain injury model

[0067] First, clean the operating table, and sterilize the required surgical instruments in advance by high-pressure steam sterilization. Prepare sterile surgical drapes, suture threads, suture needles, and povidone iodine. Fast the mice 8 hours before surgery to avoid vomiting or aspiration during the operation. Prepare 3% pentobarbital solution with distilled water and inject it intraperitoneally into the mice according to the dose standard of 30 mg / kg for anesthesia. Clamping the limbs without response indicates complete anesthesia. Then fix the mice on the trauma model device, depilate and disinfect the heads of the mice, locate at 2.0 mm behind the bregma and 2.0 mm outside the midline, incise the scalp to expose the parietal bone, and use a micro-drilling device (RWD, Shenzhen, China) to expose the brain tissue. Expand it to a bone window with a diameter of 3.5 mm while keeping the dura mater intact. Mice in the Sham group were not given traumatic brain injury treatment after the bone window was opened, and the surgical incision was sutured layer by layer with sterile suture threads, and disinfected with povidone iodine cotton balls. 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, residence time: 200 ms). The cleaning, suture, and disinfection steps and operations were the same as those in the Sham group. Place the mice on a heating blanket for 2 - 3 hours, and place them in a breeding cage after they wake up (mice can freely obtain food and water in the cage).

[0068] 1.4 Pre-experiment verification in the traumatic brain injury model

[0069] Mice in the Sham group and the TBI group were sacrificed by intraperitoneal injection of 3% pentobarbital solution on the 3rd and 7th days after traumatic brain injury. The brain tissue at the injury site was taken for qPCR to detect the expression level of METTL1 mRNA.

[0070] The qPCR results showed that, Figure 3, compared with the sham operation group (Sham group), the mRNA level of METTL1 in the TBI group of mice gradually increased on the 3rd and 7th days after traumatic brain injury.

[0071] Example 2: In vivo functional experiment after knocking down METTL1

[0072] The construction of cell-specific METTL1 knockdown adeno-associated virus type 9 (AAV9) was completed by Vigene Biosciences Co., Ltd. sh-METTL1 group: vector AAV9-gfaABC1D-GFP-mir30-shMETTL1, where gfaABC1D is an astrocyte-specific promoter, sh-METTL1 is the shRNA for knocking down METTL1, and the virus titer is 5.1×10 13 viral genomes / mL; sh-NC group: vector AAV9-gfaABC1D-GFP-mir30 inserted with a nonsense sequence, and the virus titer is 4.6×10 13 viral genomes / mL.

[0073] Among them, the nucleotide sequence of gfaABC1D is shown in SEQ ID NO.1;

[0074] The nucleotide sequence of sh-METTL1 is shown in SEQ ID NO.2.

[0075] 2.1 In vivo functional experiment of spinal cord injury model after knocking down METTL1

[0076] AAV9 of the sh-METTL1 group and the sh-NC group was injected into the intratheca of mice using a microsyringe at a dose of 2 μL per mouse. After 3 weeks, a spinal cord injury model was established. Subsequently, the mice were divided into 4 groups: sh-METTL1+SCI group, sh-METTL1+sham group, sh-NC+SCI group, sh-NC+sham group. The recovery of hind limb motor function of the 4 groups of mice was evaluated by BMS score on the 0th, 1st, 3rd, 7th, 14th, 21st, 28th, and 35th days after spinal cord injury; on the 7th day, the mice were sacrificed after anesthesia with intraperitoneal injection of 3% pentobarbital solution, and the spinal cord tissue at the injury site was taken for qPCR to evaluate inflammatory indexes (IL-1β, IL-6, CCL2, CXCL2) and ELISA detection (IL-6, IL-1β, and CCL2); on the 35th day, the mice were sacrificed after anesthesia with intraperitoneal injection of 3% pentobarbital solution, and the spinal cord tissue at the injury site was taken for qPCR to evaluate nerve regeneration indexes (NGF, BDNF).

[0077] BMS score: It is applicable to evaluating the recovery of motor function in 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 dedicated to the hindlimb function of mice, including a main scoring system and a secondary scoring system. Compared with the BBB score, the BMS scoring system is more sensitive and reliable.

[0078] The test was operated by two experienced researchers. The BMS main score ranges from 0 points (no ankle movement) to 9 points (fully normal motor ability), and the specific scoring details are shown in Table 1. BMS behavioral assessments were performed on days 0, 1, 3, 7, 14, 21, 28, and 35 after spinal cord injury.

[0079] The specific scoring steps are as follows:

[0080] ① One day before the surgery, normal mice were placed in the open field to familiarize themselves with the environment;

[0081] ② BMS scoring started on the first day after the surgery, and the observation period for the BMS score of mice was 5 minutes;

[0082] ③ After recording the video with a video recorder, it was analyzed with a computer, or scores could be given immediately after being proficient in the scoring details;

[0083] ④ Result analysis: The level of the BMS score represents the state of the recovery of the hindlimb motor function in mice after spinal cord injury. The higher the score, the better the recovery. When it reaches 9 points, it is equivalent to that of normal animals.

[0084] Table 1. BMS Scoring Details

[0085]

[0086] Annotation:

[0087] Slight: Activity less than half;

[0088] Extensive: Activity greater than half;

[0089] Normal and strong placement of the lower limbs: Actively place the claws on the ground, and both the big toe and the last toe are in contact with the ground;

[0090] Supporting gravity: The hindlimbs must be lifted high enough so that the root of the tail is lifted off the ground, and the knees do not touch the ground during walking;

[0091] Stepping and walking: The hindlimbs can support the body weight when initially leaving the ground, step forward, and then support the body weight again when touching the ground;

[0092] Good coordination: Each step of the forelimb is accompanied by a step of the hindlimb, and the hindlimbs alternate in stepping. To be able to evaluate, the mouse must step continuously and uniformly and at least three times its own body length. Pauses or hesitations during walking cannot be included. To detect coordination, at least three times are required. If one of the three attempts is unsuccessful, it is considered uncoordinated;

[0093] Claw position: The claws are placed parallel to the body. If they move outwards away from the body, it is external rotation, and if they move inwards towards the body, it is internal rotation;

[0094] Severe trunk instability: During monitoring, obvious postural instability is shown in the hindlimbs, such as extreme tilting, obvious swaying, and approaching falling.

[0095] The qPCR results showed that, such as Figure 4 、 Figure 5 、 Figure 6 and Figure 7 , in the acute stage of spinal cord injury (7 days), compared with the control group (sh-NC+SCI group), the inflammation-related indicators in the spinal cord tissue of the experimental group (sh-METTL1+SCI group) mice were significantly reduced. The ELISA results were as Figure 8 、 Figure 9 and Figure 10 showed that the inflammation-related indicators in the experimental group were significantly reduced. And in the chronic stage of spinal cord injury (35 days), as Figure 11 and Figure 12 of the qPCR results showed that the levels of various neurotrophic factors were significantly increased. The BMS score results also showed that, as Figure 13 showed, the improvement of the hindlimb motor function of the experimental group mice was more obvious. The above results suggest that specific knockdown of METTL1 in mouse astrocytes significantly reduces the inflammatory response caused by spinal cord injury and promotes nerve regeneration.

[0096] 2.2 In vivo functional experiments on the traumatic brain injury model after knocking down METTL1

[0097] AAV9 of the sh-METTL1 group and the sh-NC group was injected into the mouse intrathecal space using a microsyringe, and the injection dose was 2 μL / mouse. Three weeks later, a traumatic brain injury model was established. Subsequently, the mice were divided into 4 groups: sh-METTL1+TBI group, sh-METTL1+sham group, sh-NC+TBI group, sh-NC+sham group. The above 4 groups of mice were sacrificed after being anesthetized with 3% pentobarbital solution by intraperitoneal injection on the 7th day, and the brain tissue at the injury site was taken for ELISA detection (IL-6, IL-1β); on the 35th day, they were sacrificed after being anesthetized with 3% pentobarbital solution by intraperitoneal injection, and the brain tissue at the injury site was taken for qPCR to evaluate the nerve regeneration indicators (NGF, BDNF).

[0098] ELISA results showed that, as Figure 14 and Figure 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 tissues of mice in the experimental group (sh-METTL1 + TBI group) were significantly reduced. The qPCR results showed that, as Figure 16 and Figure 17 , the levels of various neurotrophic factors were significantly increased in the chronic phase of traumatic brain injury (35 days). The above results suggest that specific knockdown of METTL1 in mouse astrocytes significantly reduces the inflammatory response caused by traumatic brain injury and promotes nerve regeneration.

[0099] In summary, it can be generalized that knocking down METTL1 in astrocytes in vitro can reduce the inflammatory response after central nervous system injury, promote nerve regeneration and motor function recovery.

[0100] Example 3: In vitro functional experiment after knocking down METTL1

[0101] Take neonatal mice within 7 days after birth. Under sterile conditions, cut off the heads of the neonatal mice. Use fine scissors to cut open the skin and skull along the midline, taking care not to damage the cerebral cortex. Then, under a stereomicroscope, use fine forceps to strip the bilateral skulls, and then remove the entire brain below the olfactory bulb and place it in pre-cooled HBSS (Hank's balanced salt solution). Remove the meninges covering between the skull and the brain parenchyma, and cut the remaining tissue into small pieces as much as possible with forceps / scissors. Add 0.125% trypsin and DNase, and digest in a 37 °C constant temperature water bath for 15 min, shaking once every 5 min. Terminate the digestion with DMEM complete medium, centrifuge at 4 °C and 1000 rpm for 5 min, resuspend and filter to make a single-cell suspension. Inoculate the single-cell suspension into a 10 cm cell culture dish containing DMEM complete medium (DMEM, 10% FBS, 1% double antibody), and perform differential adhesion in a 37 °C incubator for 1 hour. After 1 hour, transfer the upper layer of the medium in the culture dish to a 25T culture flask pre-coated with PDL (polylysine) to obtain primary astrocytes (Astrocyte, AST).

[0102] 3.1. Detection of inflammation-related indicators:

[0103] After culturing the cells for 5-6 days, they were seeded 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 grew to 50%-60% confluence using DMEM complete medium, siRNA transfection was performed. After culturing for another 24 hours, the TNF-α group was given TNF-α protein (working concentration: 10 ng / mL), and the control group (CON group) was given an equal volume of PBS solution. After culturing for another 12 hours, the cells were harvested, and mRNA was extracted for qPCR experiments to detect inflammatory indicators (IL-1β, IL-6, CCL2, CXCL2).

[0104] Among them, the siRNA nucleotide sequence of the Si-METTL1 group is shown in SEQ ID NO.3;

[0105] The siRNA of the Si-NC group is a nonsense sequence, and its nucleotide sequence is shown in SEQ ID NO.4.

[0106] The qPCR results showed that, as Figure 18 、 Figure 19 、 Figure 20 and Figure 21 , in primary astrocytes cultured in vitro, compared with the control group (Si-NC TNF-α group), the inflammation-related indicators in the experimental group (Si-METTL1 TNF-α) were significantly reduced.

[0107] 3.2. Detection of NF-κB pathway activation:

[0108] After culturing the cells for 5-6 days, they were seeded into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. When the cells grew to 50%-60% confluence using DMEM complete medium, 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 culturing for another 24 hours, the cells were starved in serum-free medium for 6 hours, and then TNF-α (working concentration: 10 ng / mL) was added to each well to stimulate the cells. Cell proteins were extracted at the 0 min, 15 min, 30 min, and 60 min after adding TNF-α, and the related indicators of the NF-κB pathway were detected by Western Blot.

[0109] The Western Blot detection results showed that, as Figure 22 , knocking down METTL1 significantly reduced the expression level of p-p65, while the p65 level remained basically unchanged, confirming that knocking down METTL1 could inhibit the activation of the NF-κB pathway.

[0110] Example 4: Finding the relationship between METTL1 and m 7 G methylation and verification:

[0111] 4.1. Detection of m 7 G level after knocking down METTL1 in cells

[0112] The extraction method of primary astrocytes is as described in Example 3. After culturing the cells for 5 - 6 days, they were seeded into 6 - well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. When the cells grew to 50% - 60% confluence using DMEM complete medium, siRNA transfection was carried out. The control group was Si - NC, and the experimental group was Si - METTL1. The corresponding siRNA was the same as in Example 3. After continuing to culture for 24 hours, mRNA was extracted and the mRNA concentration was determined. The mRNA concentration was adjusted uniformly using sterile and enzyme - free water in sequence, and then the mRNA was diluted to 400 ng / μL, 200 ng / μL and 100 ng / μL respectively for Dot Blot to detect m 7 G methylation level, and the membrane stained with methylene blue (MB) was used as a control.

[0113] The Dot Blot results showed that, as Figure 23 , knocking down METTL1 could significantly reduce the level of m 7 G methylation.

[0114] 4.2. Detection of METTL1 content and m 7 G level under inflammatory conditions in vitro cells

[0115] The extraction method of primary astrocytes is as described in Example 3. After culturing the cells for 5 - 6 days, they were seeded into 6 - well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. When the cells grew to 50% - 60% confluence using DMEM complete medium, induction was carried out. TNF - α (working concentration 10 ng / mL) was added to each well to stimulate the cells. At the 0h, 2h, 4h, 6h, 12h, 24h after adding TNF - α, the cells were harvested to extract mRNA for qPCR experiment to detect the mRNA content of METTL1, and mRNA was extracted for DotBlot to detect m 7 G methylation level, and the membrane stained with methylene blue (MB) was used as a control.

[0116] The qPCR results showed that, as Figure 24 , the mRNA expression level of METTL1 increased with the prolongation of TNF - α stimulation time. The DotBlot results showed that, as Figure 25 , m 7 G methylation level increased with the prolongation of TNF - α stimulation time. It was inferred that under in vitro inflammatory conditions, the content of METTL1 and m7 The m6A methylation level increased synchronously.

[0117] 4.3 Detection of the content of METTL1 and m6A 7 level in vivo after spinal cord injury

[0118] The mouse spinal cord injury model was established as in Example 1. The mice in the Sham group and the SCI group were sacrificed after being anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 7th day after spinal cord injury. The spinal cord tissue at the injury site was taken to extract mRNA for Dot Blot to detect the m6A 7 methylation level, and the membrane stained with methylene blue (MB) was used as a control.

[0119] The Dot Blot results showed that, as Figure 26 shown, the m6A 7 methylation level in the SCI group of mice increased after spinal cord injury. Combining with what was described in Example 1, the mRNA level of METTL1 in the SCI group of mice gradually increased after spinal cord injury. It was thus inferred that the content of METTL1 and the m6A 7 methylation level increased synchronously after spinal cord injury.

[0120] In summary, the present invention confirmed that knocking down METTL1 could reduce the m6A 7 methylation level, and verified that the changes in the content of METTL1 and the m6A 7 methylation level were synchronous both in vitro cell experiments and after spinal cord injury in vivo.

[0121] The above are only the embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. Use of an inhibitor of METTL1 in the preparation of a medicament for treating inflammation after central nervous system injury; The central nervous system injury is spinal cord injury or traumatic brain injury; The inhibitor of METTL1 is siRNA that interferes with the expression of METTL1, or a virus packaged with shRNA that interferes with the expression of METTL1, wherein, The nucleotide sequence of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the shRNA is as shown in SEQ ID NO.

2.

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

3. The application according to claim 1, wherein The inhibitor of METTL1 reduces the m 7 G methylation level by inhibiting the expression of METTL1, thereby playing a role in inhibiting inflammation.

4. The application according to claim 1, 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 as shown in SEQ ID NO.1; shMETTL1 is an shRNA that interferes with the expression of METTL1.

5. Use of an inhibitor of METTL1 in the preparation of a medicament for promoting nerve regeneration after central nervous system injury; The central nervous system injury is spinal cord injury or traumatic brain injury; The inhibitor of METTL1 is siRNA that interferes with the expression of METTL1, or a virus packaged with shRNA that interferes with the expression of METTL1, wherein, The nucleotide sequence of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the shRNA is as shown in SEQ ID NO.

2.

6. The application according to claim 5, wherein The inhibitor of METTL1 can promote the expression of neurotrophic factors by inhibiting the expression of METTL1, and thus promote nerve regeneration.

7. 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, Comprising an inhibitor of METTL1, the inhibitor of METTL1 is an siRNA that interferes with the expression of METTL1, or a virus packaged with an shRNA that interferes with the expression of METTL1, wherein the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2; The central nervous system injury is spinal cord injury or traumatic brain injury.

8. The drug according to claim 7, 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 as shown in SEQ ID NO.1; shMETTL1 is an shRNA that interferes with the expression of METTL1.

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