Application of TRIM56 inhibitor in treatment of cerebral ischemic diseases

Abstract

The invention provides an application of a TRIM56 inhibitor in treatment of cerebral ischemic diseases, explores TRIM56 as a potential target gene for preventing and / or treating cerebral ischemic diseases by verifying a mutual relationship between TRIM56 expression and cerebral ischemia, provides an application of TRIM56 in preparation of drugs for preventing and / or treating cerebral ischemic diseases, provides a new application of TRIM56 as a treatment target, and provides a new application of TRIM56 in preparation of drugs for preventing and / or treating cerebral ischemic diseases and a new application of TRIM56 in preparation of drugs for preventing and / or treating cerebral ischemic diseases. And theoretical basis and practical support are provided for research and development of cerebral ischemia drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of cerebral ischemia drugs, and in particular to application of a TRIM56 inhibitor in treating cerebral ischemic diseases. Background Art

[0002] Cerebrovascular disease is one of the diseases with high morbidity and mortality worldwide, especially cerebral ischemic diseases, including acute ischemic stroke. According to statistics from the World Health Organization, the mortality rate of stroke ranks among the top causes of death from diseases worldwide. It is estimated that by 2030, the number of stroke-related deaths worldwide will reach about 8.6 million, accounting for more than 6% of all deaths. Among them, cerebral ischemia, especially ischemia-reperfusion injury (OGD / R, oxygen / glucose deficiency and reperfusion), is an important cause of neurological damage and long-term neurodegenerative diseases. Acute ischemic stroke and chronic ischemia-induced neural damage directly affect the patient's quality of life and may lead to long-term neurological dysfunction, which has serious clinical significance. Therefore, studying the molecular mechanism of cerebral ischemia, finding new targets and developing effective treatments have become hot topics in the current field of cerebrovascular diseases.

[0003] The mechanism of cerebral ischemic injury is very complex, involving the activation and regulation of multiple cell signaling pathways. Ischemic injury usually induces pathological processes such as inflammatory response, oxidative stress, and cell death in brain tissue. Studies have shown that neurons can be damaged through multiple pathways such as oxidative stress and calcium ion overload during ischemia, ultimately leading to cell death and dysfunction. In addition, the destruction of the blood-brain barrier, the excessive release of inflammatory factors, and the activation of microglia are also considered to be important factors in secondary injury after stroke. These pathological changes not only lead to neuronal death, but may also cause irreversible damage to brain tissue.

[0004] TRIM56 (TNF receptor-associated regulatory protein 56) is a protein that plays an important role in immune response and belongs to the TRIM (Tripartite Motif) protein family. Studies have found that TRIM56 can regulate intracellular inflammatory responses by interacting with other molecules and play an important regulatory role in a variety of diseases. Specifically, TRIM56 regulates immune responses and cellular stress responses by interacting with signaling pathways such as NF-κB and IRF3. Excessive activation or inhibition of TRIM56 is closely related to the occurrence of a variety of immune-related diseases, inflammatory responses, and tumors. However, the role of TRIM56 in cerebral ischemic injury has not been systematically studied, although studies have shown that TRIM56 may play a role in neurological diseases by affecting inflammatory responses and immune regulation.

[0005] Early studies have found that TRIM56 expression is elevated in some neurodegenerative diseases and acute brain injury, and may affect the pathological process by regulating neuroinflammatory responses and cell death pathways. For example, in animal models of ischemic stroke, changes in TRIM56 expression are correlated with the severity of neural damage, suggesting that TRIM56 may be a key regulator in cerebral ischemia-reperfusion injury. Further research is expected to reveal the specific mechanism of TRIM56 in ischemic brain injury and provide new molecular targets for the treatment of cerebral ischemia-related diseases.

[0006] In summary, although the role of TRIM56 in immune response and inflammation has been widely studied, its specific mechanism of action in cerebral ischemic injury has not yet been clarified. Exploring the role of TRIM56 in cerebral ischemia models and revealing its mechanism of regulating neural injury may provide a theoretical basis for the development of new stroke treatment strategies. Summary of the invention

[0007] The purpose of the present invention is to clarify the relationship between the expression of TRIM56 and cerebral ischemia, and to provide the application of TRIM56 in screening drugs for protecting brain function and / or preventing, alleviating and treating cerebral ischemia-related diseases (such as ischemic brain injury, stroke, etc.). By exploring TRIM56 as a potential target gene for protecting brain function and / or preventing, alleviating and treating cerebral ischemia, the purpose is to discover new uses of TRIM56 and provide theoretical basis and practical support for the development of drugs for cerebral ischemia.

[0008] The technical solution of the present invention is achieved in this way: The first aspect of the present invention is to propose the use of a TRIM56 inhibitor in the preparation of a drug for preventing or treating cerebral ischemic diseases.

[0009] Furthermore, the TRIM56 inhibitor includes siRNA, shRNA or antisense RNA that inhibits the expression of TRIM56.

[0010] Furthermore, the TRIM56 inhibitor is a viral expression vector containing shRNA.

[0011] Preferably, the viral expression vector is an adenovirus or a lentivirus.

[0012] Furthermore, the nucleotide sequence targeted by the shRNA is GCTTCCTCTGTCAACCTTGTT.

[0013] Furthermore, the cerebral ischemic disease includes ischemic brain injury and cerebral stroke.

[0014] Furthermore, the drug is used for at least one of the following purposes: a) Improve brain tissue edema and reduce infarct volume; b) Improve brain damage during cerebral ischemia-reperfusion injury; c) inhibit neuronal inflammatory response caused by cerebral ischemia; d) Inhibit neuronal inflammatory response induced by cerebral ischemia-reperfusion.

[0015] Furthermore, in the use, the inhibition of inflammatory response in c) and / or d) includes inhibiting at least one of TNF, IL6, IL1B, CCL2, and CXCL2.

[0016] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0017] Furthermore, the drug is used for intravenous injection.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The present invention verifies the relationship between the expression of TRIM56 and cerebral ischemia, explores TRIM56 as a potential target gene for preventing and / or treating cerebral ischemic diseases, and provides the application of TRIM56 in the preparation of drugs for preventing and / or treating cerebral ischemic diseases, provides a new use of TRIM56 as a therapeutic target, and provides a theoretical basis and practical support for the research and development of drugs for cerebral ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Figure 1 shows the expression of TRIM56 in the brain ischemia model described in Example 1; Figure A shows the expression of TRIM56 in the brain tissue of mice after 18h, 24h, and 48h of ischemia-reperfusion in sham surgery (Sham) and transient middle cerebral artery occlusion surgery (t / MCAO). The results show that the expression of TRIM56 is significantly increased in the brain ischemia mouse model. 18h, 24h, and 48h represent 18 hours, 24 hours, and 48 hours (**: p <0.01 vs wild-type mouse Sham group); Figure B is the mRNA and protein expression of TRIM56 in Sprague-Dawley rat primary neurons (CCNs) and OGD / R-induced brain ischemia model. The results show that in the brain ischemia cell model, the mRNA level and protein level of TRIM56 were significantly increased in the OGD / R-induced group (**: p<0.01 vsControl group).

[0021] Figure 2 The results of the effects of TRIM56 knockout and TRIM56 overexpression on the brain ischemia model described in Example 2. Figure A shows the Berderson nine-point scale neurological function scores of wild-type (WT) mice and TRIM56 knockout (KO) mice 24 hours after t / MCAO surgery. The results show that in the brain ischemia model, the knockout of TRIM56 can significantly protect brain function and reduce brain damage (##: p <0.01 vs WT-24h group); Figure B is the brain dissection of wild-type (WT) mice and TRIM56 knockout (KO) mice 24h after t / MCAO surgery, and the TTC method was used to evaluate brain infarction and edema. The results of TTC staining and quantitative analysis showed that brain tissue edema and infarction volume in the KO group (24h) were significantly improved (##: p <0.01;#: p <0.05 vs WT-24h group); Figure C is the H&E staining results of wild-type (WT) mice and TRIM56 knockout (KO) mice 24 hours after Sham or t / MCAO surgery. H&E results further confirmed that the knockout of TRIM56 significantly improved brain damage during cerebral ischemia-reperfusion injury; Figure D shows the CD11b immunofluorescence staining results of wild-type (WT) mice and TRIM56 knockout (KO) mice 24 hours after Sham or t / MCAO surgery, as well as the effects on the expression levels of inflammatory response markers. The results showed that the knockout of TRIM56 significantly inhibited the inflammatory response caused by cerebral ischemia (**: p <0.01;*: p <0.05 vs WT-Sham group; ##: p <0.01;#: p <0.05 vs WT-24h group).

[0022] Figure 3 These are the experimental results of control (AAV9-Ctrl) mice and TRIM56 overexpression (AAV9-TRIM56) mice 24 hours after Sham or t / MCAO surgery; the results obtained are opposite to those of TRIM56-KO, further confirming that the knockout of TRIM56 can significantly improve brain damage during cerebral ischemia-reperfusion injury (**: p <0.01;*: p <0.05; ns: no significant difference vs AAV9-Ctrl-24h group).

[0023] Figure 4Figure 1 shows the effects of TRIM56 interference adenovirus and overexpression adenovirus on the inflammatory response of neurons in the brain ischemia model. Figure A shows the effects of Sprague-Dawley rat primary neurons (CCNs) infected with adenovirus AdshRNA and AdshTRIM56 on the expression levels of inflammatory response markers in the OGD / R-induced brain ischemia model. The results showed that the knockdown of TRIM56 significantly inhibited the inflammatory response induced by OGD / R (**: p <0.01;*: p <0.05 vsAdshRNA-OGD / R group); Figure B shows the effect of Sprague-Dawley rat primary neurons (CCNs) infected with adenovirus AdGFP and AdTRIM56 on the expression levels of inflammatory response markers in the OGD / R-induced brain ischemia model. The results showed that overexpression of TRIM56 significantly promoted the inflammatory response induced by OGD / R (**: p <0.01;*: p <0.05 vsAdGFP-OGD / R group). DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In one embodiment, the relationship between TRIM56 expression and brain defect is determined by experiment: 1. In the case of brain ischemia, TRIM56 protein expression is significantly upregulated Transient Middle Cerebral Artery Occlusion (t / MCAO) was used to establish a mouse brain ischemia model, extract mouse brain tissue protein and RNA, and perform Real-Time PCR (Real-time Polymerase Chain Reaction) and SDS-PAGE-Western blot (WB) to analyze the changes in TRIM56 mRNA and protein levels in the in vivo brain ischemia model. The results showed that in the in vivo model, both mRNA and protein levels were significantly increased ( Figure 1 A).

[0026] Oxygen-Glucose Deprivation / Reperfusion (OGD / R) was used to induce brain ischemia model of primary cortical neurons of Sprague-Dawley rats. Protein and RNA were extracted from primary cortical neurons of Sprague-Dawley rats. Real-time PCR (Real-time Polymerase Chain Reaction) and SDS-PAGE-Western blot (WB) were performed to analyze the changes in mRNA and protein levels of TRIM56 in the in vitro brain ischemia model. The results showed that in the in vitro model, both mRNA and protein levels were significantly increased ( Figure 1 B).

[0027] 2. In vivo, TRIM56 knockout (KO) improved brain necrosis on the ischemic side of the brain; TRIM56 overexpression (AAV9) aggravated brain necrosis on the ischemic side of the brain To further explore the effect of TRIM56 on brain ischemia, we constructed TRIM56 knockout (KO) mice and TRIM56 overexpression (AAV9) mice, and performed t / MCAO surgery together with wild-type WT and AAV9-Ctrl control mice to induce MCAO model. After 24 hours of reperfusion, neurological function was evaluated using the Berderson nine-point scale. Compared with the wild-type WT 24-hour group, the KO 24-hour group showed lower neurological scores ( Figure 2 A). After brain tissue dissection, TTC staining was used to evaluate cerebral infarction and edema. TTC staining and quantitative analysis results showed that the KO 24-hour group had smaller edema and infarction volume ( Figure 2 B). H&E staining results further confirmed that TRIM56 deficiency improved the necrosis of ischemic brain tissue ( Figure 2 C). In addition, immunofluorescence assay and Real-Time PCR showed that the inflammatory response in KO mice was significantly improved ( Figure 2 D). The results obtained in TRIM56 overexpression (AAV9) mice were opposite to those in the KO group ( Figure 3 ). These results indicate that loss of TRIM56 significantly ameliorates brain damage during cerebral ischemia.

[0028] 3. In vitro, TRIM56 interference adenovirus inhibited the inflammatory response of neurons in the brain ischemia model; TRIM56 overexpression adenovirus promoted the inflammatory response of neurons in the brain ischemia model Sprague-Dawley rat primary neurons (CCNs) were isolated and cultured in vitro, and TRIM56 interference (Adsh TRIM56) and overexpression (Ad TRIM56) adenoviruses were constructed. AdshRNA and AdGFP were used as control plasmids, respectively, and the OGD / R-induced neuronal brain ischemia model was used to study the regulatory effect of TRIM56 on the neuronal brain ischemia model. The relationship between TRIM56 and brain ischemia was determined through experiments: TRIM56 knockdown mediated by interfering RNA (shRNA) adenovirus inhibited the inflammatory response of neurons in the brain ischemia model ( Figure 4 A) Adenovirus-mediated overexpression of TRIM56 promotes neuronal inflammatory responses in the brain ischemic model ( Figure 4 B).

[0029] According to the results of the above examples, the protein expression of TRIM56 is significantly upregulated when cerebral ischemia occurs. TRIM56 gene deletion significantly improved brain tissue necrosis in the cerebral ischemia model, while TRIM56 overexpression aggravated brain tissue damage. It can be seen that TRIM56 plays an important role in the process of cerebral ischemia and may affect the development of brain damage by regulating inflammatory responses. Therefore, the potential role of the TRIM56 gene in cerebral ischemia and related diseases deserves further exploration, which may provide new targets for future clinical treatment.

[0030] Based on the above functions of TRIM56 in cerebral ischemia, an application of TRIM56 is provided, which is mainly reflected in the application of TRIM56 inhibition in protecting brain function and alleviating cerebral ischemia damage, especially the application of TRIM56 inhibition in the preparation of drugs for protecting brain function and / or preventing, alleviating and / or treating cerebral ischemia-related diseases.

[0031] Unless otherwise specified, the materials and reagents used are commonly used in the art, and the experimental methods used are well-known to those skilled in the art.

[0032] Experimental animals and feeding Experimental animals: For animal experiments, 7-9 weeks old, weighing 25-27 g, with a C57BL / 6J background were used as experimental subjects; for cell experiments, 1-day-old Sprague-Dawley rat pups were used as experimental subjects.

[0033] Feeding environment: SPF rat feed was purchased from Beijing Huafukang Biotechnology Co., Ltd. Feeding conditions: room temperature between 22-24°C, humidity between 40-70%, alternating light and dark lighting time of 12h, free access to water and food.

[0034] Example 1 Detection of TRIM56 mRNA and protein levels in the OGD / R-induced Sprague-Dawley rat primary neurons (CCNs) brain ischemia model 1. Primary Neuron Culture of Newborn Sprague-Dawley Rat Sucklings First, one-day-old Sprague-Dawley rats were selected and euthanized. Then, the rat brain was carefully dissected and the cerebral cortex was removed. The cerebral cortex tissue was placed in a culture dish containing 1× phosphate buffered saline (PBS) and the tissue was sheared under a microscope to remove blood vessels, meninges, and other non-neural tissues.

[0035] Next, the cerebral cortex tissue was digested at 37°C for 15 minutes using 0.125% trypsin (Trypsin, GIBCO) solution. After digestion, a buffer containing 10% fetal bovine serum (FBS, GIBCO) and 1% DNase (Roche) was added to terminate the digestion process. The digested cell suspension was filtered through a 40 μm cell sieve to remove undigested tissue blocks. The filtered cell suspension was centrifuged at 1500 rpm for 10 minutes to collect the cell pellet.

[0036] The collected cell pellet was resuspended in complete DMEM / F-12 medium containing 10% FBS, and then the cells were seeded into culture dishes or culture flasks coated with poly-L-lysine (10 mg / mL, Sigma) and incubated at 37°C with 5% CO 2 After 3 hours of culture, the culture medium was replaced with Neurobasal-A medium (GIBCO) supplemented with 2% B27 supplement (GIBCO), and the culture medium was changed every 48 hours.

[0037] 2. Establishment of OGD / R-induced neuronal brain ischemia model For OGD / R (oxygen-glucose deprivation / reperfusion) treatment, the neuronal culture medium was first replaced with glucose-free DMEM (11966025, GIBCO), and the cells were placed in 95% N 2 and 5% CO 2 After treatment, the culture medium was replaced with normal culture medium and the cells were transferred to a hypoxic incubator containing 21% O 2 and 5% CO 2 The cells in the control group were cultured in the presence of normal glucose and fetal bovine serum (FBS) and in a humidified atmosphere containing 95% air and 5% CO for the same period of time. 2 in the cultivation environment.

[0038] 3. Analysis of TRIM56 mRNA and protein levels The induced neurons were collected, total RNA and total protein were extracted, and Real-Time PCR and Western blot were used to detect the mRNA and protein levels of TRIM56 in the brain ischemic model. Figure 1 As shown in B, after OGD / R induction, the mRNA level and protein level of TRIM56 were significantly higher than those in the neurons induced by the Control group. Figure 1 The bar graph on the right side of B is the protein quantitative data statistics of the right side of the figure, which can intuitively reflect that the level of TRIM56 protein is significantly higher than that of neurons induced by the Control group. ( Figure 1 In B, the Control group is the control group; the OGD / R group is the experimental group, and the neurons induced by OGD / R will establish their brain deficiency model; TRIM56 is the target gene antibody used to detect protein expression in this sample; GAPDH is the protein standardization internal reference antibody used to determine whether the protein amount of each band is consistent. ) 3.1 Western blot Western blot was used to detect the target gene antibody (TRIM56) (1) Neuronal protein extraction: aspirate the primary neurons cultured on the wall and discard the culture medium, resuspend the precipitate with protein lysis buffer containing 1% SDS, mix well and boil at 95 degrees for 15 minutes; (2) Protein quantification and protein sample preparation: Take 4 µL of the lysed protein sample and dilute it to 60 µL for protein quantification using the BCA protein quantification kit (Thermo Company, Catalog No. 23227); (3) SDS-PAGE gel electrophoresis: After preparing 10% SDS-PAGE gel in advance, add the corresponding electrophoresis solution into the electrophoresis tank and perform protein electrophoresis; (4) Transfer: Use PVDF membrane and add transfer solution to transfer membrane; (5) Blocking: After transfer, remove the PVDF membrane and wash it twice with TBST, then add 5% skim milk for blocking and incubate at room temperature for 1 h. (6) Primary antibody incubation: discard the blocking solution, wash three times with TBST, add the corresponding primary antibody, and incubate on a shaker at 4°C overnight; (7) Secondary antibody incubation: The next day, after the primary antibody was recovered, the membrane was washed three times with TBST and then the corresponding secondary antibody prepared with 5% skim milk was added and incubated for 1 h. (8) Development: After the secondary antibody incubation is completed, wash the membrane three times with TBST and then develop it using a gel imaging system.

[0039] 3.2 Real-Time PCR Experiment Real-Time PCR was used to detect the primers of genes related to inflammatory response markers (TNF, IL6, IL1B, CCL2, CXCL2) (1) Extraction of RNA from primary neurons: Take the attached cultured primary neurons and add 500 µL Trizol (Sigma-Aldrich, catalog number 9424-100 mL) / well (6-well plate). Blow off the cells and transfer to a 1.5 mL centrifuge tube. Add 100 µL / tube of chloroform and shake vigorously to mix. Then let it stand for 10 min. After standing, place it in a low-temperature centrifuge at 12,000 rpm for 10 minutes. Then remove the top aqueous phase liquid, transfer it to a new centrifuge tube, add an equal volume of isopropanol, mix and let it stand for 10 min. Then place it in a low-temperature centrifuge at 12,000 rpm for 10 minutes. After centrifugation, discard the supernatant and keep the precipitate. Wash the precipitate with 75% ethanol. Repeat three times. Dry the precipitate, add DEPC water to dissolve the precipitate and measure the RNA concentration. (2) RNA reverse transcription: According to the instructions of the reverse transcription kit (Vazyme, catalog number R323-01), 2 µg of RNA was taken for RNA reverse transcription to construct a cDNA library; (3) Real-time fluorescence quantitative PCR analysis: After the cDNA obtained by reverse transcription is used as a template and the corresponding primers and qPCR working solution are added to prepare the qPCR system, it is added to a 96-well or 384-well plate for real-time fluorescence quantitative PCR analysis.

[0040] Example 2 Pathological status of TRIM56 in the mouse brain ischemia model after transient middle cerebral artery occlusion (t / MCAO) 1. The brain ischemia model uses transient middle cerebral artery occlusion surgery (t / MCAO). The model operation process is as follows: 1.1 Preoperative preparation Anesthesia: First weigh the mouse, calculate the amount of anesthetic (3% sodium pentobarbital) required according to 90 mg / kg body weight, inject it intraperitoneally, and record the injection time. Evaluate the anesthetic effect after 2 to 3 minutes. The standard for successful anesthesia is that there is no obvious reaction to clamping the tail or toes and the mouse is in good condition (generally there is no obvious reaction about 10 minutes after injection, and the mouse responds to clamping the toes about 50 minutes after anesthesia. The best time for surgery is about 30 minutes after anesthesia).

[0041] Preparation of the surgical area: Place the index finger of your left hand on the back of the mouse's head, and the thumb and index finger on the mouse's upper limbs to expose the mouse's chest. Use a pet electric clipper to remove the mouse hair on the left chest, left chest, and left forelimb armpit. After shaving, wipe the shaved area with wet gauze to remove excess mouse hair.

[0042] Exposure and positioning of the surgical area: Fix the mouse on the operating table with its back facing up. Use an appropriate fixation device (such as an anesthesia table or rubber band) to ensure the stability of the mouse during surgery. Confirm the position of the mouse's head on the operating table, expose its skull, and expose its brain by gently pressing the skin around the mouse's neck.

[0043] Shaving and disinfection: Use pet electric clippers or fine shaving tools to shave the hair on the mouse's head, focusing on the area from the mouse's forehead to the neck, ensuring that the entire surgical area is exposed. After shaving, gently wipe the shaved area with wet gauze to remove hair residues and ensure that the surgical area is clean and free of debris.

[0044] Disinfection of surgical area: Use sterile saline to clean the shaved area to remove excess sebum and dirt. Use 75% alcohol or iodine solution to disinfect the shaved area. Wait for a while (about 1-2 minutes) after applying the disinfectant to ensure that the surgical area is sterile. After applying the disinfectant, wipe it again with wet gauze to remove excess disinfectant.

[0045] 1.2 Transient middle cerebral artery occlusion (t / MCAO) The experimental mice were anesthetized with 2% isoflurane-oxygen mixed gas, and cerebral ischemia was continuously monitored by laser Doppler blood flow meter (Perimed, Periflux System 5010). To establish the t / MCAO model, a 6-0 silicon-coated monofilament suture (Doccol Corporation, Cat#602156PK5Re) was inserted into the left external carotid artery, advanced toward the internal carotid artery, and wedged into the cerebral artery circle to block the origin of the middle cerebral artery (MCA). After 45 minutes, the monofilament suture was removed to restore blood flow reperfusion. A decrease of more than 75% in cerebral blood flow during ischemia and a recovery of more than 70% after reperfusion were considered the criteria for successful establishment of the ischemia-reperfusion model. All surgeries were performed under double-blind conditions. The experimental mice in the sham group (Sham group) did not receive ischemic treatment, but the other operation steps were the same as those in the t / MCAO group.

[0046] 1.3 Postoperative Care After transient middle cerebral artery occlusion (t / MCAO), when the mice showed spontaneous breathing and strong reaction to toe pinching, they were placed in a cage containing autoclaved bedding, feed and drinking water and continued to be raised and observed in the breeding room.

[0047] 2. Sampling: Turn on the analytical balance, adjust to zero, and then weigh and kill the mouse. Expose the skull by making a small incision in the center of the mouse's head. Using a microsurgical scalpel or fine scissors, carefully cut the skin and muscles until the skull is completely exposed. Use a microdrill or hand tool (such as a scraper) to drill a hole in the skull to expose the brain tissue, and use forceps or a scalpel to gently separate the brain tissue from the skull, making sure to minimize damage. Quickly remove the mouse brain tissue and place it on the pre-prepared sterile gauze.

[0048] 3. Gently rinse the brain surface with sterile saline to remove blood and impurities. Avoid excessive mechanical damage to the brain tissue during operation. Weigh and record, put the brain tissue into the corresponding cryopreservation tube, quickly place it in a liquid nitrogen tank, and store it in a -80℃ refrigerator for molecular biology testing.

[0049] 4. Pathological analysis of TRIM56 in brain ischemia model 24 hours after t / MCAO surgery, the neurological function scores of wild-type (WT) mice and TRIM56 knockout (KO) mice were measured by Berderson nine-point scale, and brain tissues were tested for TTC, H&E, and CD11B. The results are shown in Figure 2 shown. Figure 2 A represents the neurological function score, B, C, and D represent the results of TTC, H&E staining, and CD11B immunofluorescence staining, respectively. The results showed that the knockout of TRIM56 significantly improved brain damage during cerebral ischemia-reperfusion injury and inhibited the inflammatory response caused by cerebral ischemia.

[0050] Example 3 Effects of TRIM56 interference (AdshTRIM56) adenovirus on OGD / R-induced primary neuronal brain ischemia model Adenovirus containing TRIM56 interference (AdshTRIM56), i.e., shRNA-TRIM56 (silencing RNA-TRIM56 fusion protein) was used, and the nucleotide sequence targeted by shRNA was GCTTCCTCTGTCAACCTTGTT. AdshGFP (shRNA (silencing RNA) adenovirus targeting GFP protein, used as a control) was used to evaluate the role of TRIM56 in primary neurons of Sprague-Dawley rats. The method is as follows: 3-day cultured primary neurons of SD rats, the culture medium was discarded, and AdshGFP and AdshTRIM56 adenovirus diluted to appropriate titers (MOI = 10) were added respectively, and incubated at 37°C, 5% CO 2After 4 hours of culture, the virus supernatant was discarded and fresh culture medium was added to continue culture for 12 hours. On this basis, the ischemia-reperfusion (OGD / R) model was induced by OGD / R treatment: Hypoxia / glucose deficiency treatment: DMEM medium without glucose was replaced, all serum was removed, and cells were placed in 95% N 2 and 5% CO 2 Reperfusion treatment: After 3 hours, the cells were returned to the normal culture medium and placed back into the normal gas environment (95% air, 5% CO 2 ) for 6 hours, and then Real-Time PCR and Western blot were used to detect the mRNA and protein levels of TRIM56 in the brain ischemia model, and Real-Time PCR was used to detect the expression levels of inflammatory response markers. Figure 4 As shown in A, TRIM56 was successfully knocked down. Compared with the AdshGFP infection group, AdshTRIM56 infection significantly inhibited the inflammatory response (the above adenoviruses were constructed by Professor Li Hongliang's laboratory).

[0051] Example 4 Effects of TRIM56 overexpression (AdTRIM56) adenovirus on OGD / R-induced primary neuronal brain ischemia model AdGFP (adenovirus containing GFP (green fluorescent protein) was used as a control) and Ad TRIM56 (adenovirus containing GFP-TRIM56 (green fluorescent protein-TRIM56 fusion protein) were used to evaluate the role of TRIM56 in primary neurons of Sprague-Dawley rats. The method is as follows: 3-day-old SD rat primary neurons were cultured, the medium was discarded, and AdGFP and AdTRIM56 adenovirus diluted to an appropriate titer (MOI = 10) were added, respectively, and incubated at 37°C, 5% CO 2 After 4 hours of culture, the virus supernatant was discarded and fresh culture medium was added to continue culture for 12 hours. On this basis, the ischemia-reperfusion (OGD / R) model was induced by OGD / R treatment: Hypoxia / glucose deficiency treatment: DMEM medium without glucose was replaced, all serum was removed, and cells were placed in 95% N 2 and 5% CO 2 Reperfusion treatment: After 3 hours, the cells were returned to the normal culture medium and placed back into the normal gas environment (95% air, 5% CO 2) for 6 hours, and then Real-Time PCR and Western blot were used to detect the mRNA and protein levels of TRIM56 in the brain ischemia model, and Real-Time PCR was used to detect the expression levels of inflammatory response markers. Figure 4 As shown in B, TRIM56 was successfully overexpressed. Compared with the AdGFP infection group, AdTRIM56 infection significantly aggravated the inflammatory response (the above adenoviruses were constructed by Professor Li Hongliang's laboratory).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of TRIM56 inhibitors in the preparation of drugs for preventing or treating cerebral ischemic diseases.

2. The use according to claim 1, characterized in that The TRIM56 inhibitor includes siRNA, shRNA or antisense RNA that inhibits the expression of TRIM56.

3. The use according to claim 1, characterized in that The TRIM56 inhibitor is an adenovirus or a lentivirus containing shRNA.

4. The use according to claim 2 or 3, characterized in that The nucleotide sequence targeted by the shRNA is GCTTCCTCTGTCAACCTTGTT.

5. The use according to claim 1, characterized in that The cerebral ischemic diseases include ischemic brain injury and cerebral stroke.

6. The use according to claim 1, characterized in that The drug is used for at least one of the following purposes: a) Improve brain tissue edema and reduce infarct volume; b) Improve brain damage during cerebral ischemia-reperfusion injury; c) inhibit neuronal inflammatory response caused by cerebral ischemia; d) Inhibit neuronal inflammatory response induced by cerebral ischemia-reperfusion.

7. The use according to claim 6, characterized in that In the above uses, the inhibition of inflammatory response in c) and / or d) includes inhibiting at least one of TNF, IL6, IL1B, CCL2, and CXCL2.

8. The use according to claim 1, characterized in that The medicament includes a pharmaceutically acceptable carrier.

9. The use according to claim 1, characterized in that The drug is for intravenous injection.

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