Antisense oligonucleotides targeting ACSL4 gene and their applications

Abstract

The present invention belongs to the technical field of biology and biopharmaceuticals, and provides antisense oligonucleotides targeting ACSL4 gene and applications thereof. The oligonucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.7, and the sequences of the antisense oligonucleotides are modified with full-chain phosphorothioate, and 2'-O-methoxyethyl modifications are introduced into ribose of 5 bases at both ends, and all cytosines are modified with methylcytosine. Cell experiments and animal model experiments provided by the present invention prove that the ACSL4 antisense oligonucleotides can effectively alleviate the damage to nerve cells in the process of stroke, and the antisense oligonucleotides of the present invention, as a novel nucleic acid drug, can be used for the auxiliary treatment of stroke, and have broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and biopharmaceuticals, and specifically relates to an antisense oligonucleotide targeting an ACSL4 gene and an application thereof. Background Art

[0002] Stroke, also known as apoplexy, is an acute cerebrovascular disease. It refers to a disease in which the blood supply to the brain is interrupted due to ischemia or hemorrhage of the cerebral blood vessels, which eventually causes brain tissue damage. Ischemic stroke (IS) accounts for about 85% of the total number of strokes. At present, the research on anti-IS drugs mainly involves thrombolysis and neuroprotection. Clinical studies have shown that dissolving thrombi within 4.5 hours after the onset of ischemia to restore blood perfusion can save dying brain nerve cells. If used after 4.5-6 hours, pathological damage will be aggravated due to ischemia-reperfusion injury, and the mortality rate will increase. Therefore, the use of thrombolytic drugs has a strict time window limit. Neuroprotection was once considered the hope for the treatment of IS, but to date, no independent neuroprotectant has been supported by clinical efficacy. Therefore, it is extremely important to find effective treatment strategies and drugs.

[0003] In recent years, studies have found that the ferroptosis pathway mediates the death of neurons during the injury process of ischemic stroke. Ferroptosis is a new mode of cell death discovered in 2012. It is mainly triggered by the toxic accumulation of lipid peroxides on the cell membrane, which reacts with polyunsaturated fatty acids (PUFAs) and destroys the integrity of the cell membrane by inhibiting the antioxidant defense system and the accumulation of iron-dependent reactive oxygen species (ROS). The hallmark feature of ferroptosis is the peroxidation accumulation of polyunsaturated fatty acids (PUFA) in the plasma membrane. Ferroptosis is one of the most important cell death modes in cerebral ischemia-reperfusion injury and has been shown to be involved in the process of cerebral ischemia-reperfusion. Therefore, ferroptosis after IS is a potential therapeutic target against IS.

[0004] The full name of the ACSL4 gene is Acyl-CoASynthetaseLong-ChainFamilyMember4. This gene encodes an enzyme that catalyzes the combination of long-chain fatty acids and coenzyme A (CoA) to form long-chain fatty acyl-CoA. Long-chain fatty acyl-CoA is a key intermediate in lipid metabolism and biomembrane synthesis. ACSL4 increases the content of cell membrane PUFAs by catalyzing the production of fatty acyl-CoA, thereby increasing the sensitivity of cells to ferroptosis. In addition, the expression level of ACSL4 may affect the activity of the intracellular antioxidant defense system, including antioxidants such as glutathione (GSH) and related enzyme systems. When the antioxidant defense system is inhibited, PUFA peroxidation in the membrane cannot be effectively inhibited, thereby exacerbating ferroptosis. Therefore, the expression and activity of ACSL4 are closely related to the sensitivity of ferroptosis. Highly expressed or active ACSL4 increases the susceptibility of cells to ferroptosis, while inhibiting the expression or activity of ACSL4 can protect cells from ferroptosis. In view of this, the development of inhibitory drugs for ACSL4 is of great significance for preventing neurological damage in ischemic stroke.

[0005] To this end, those skilled in the art have proposed antisense oligonucleotides targeting the ACSL4 gene and applications thereof to solve the problems raised by the background technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides antisense oligonucleotides targeting ACSL4 gene and applications thereof, with the aim of providing an ACSL4 protein expression inhibitor with low toxicity and good specificity.

[0007] Another object of the present invention is to provide a drug for preventing nerve death during stroke.

[0008] To achieve the above object, the present invention provides an antisense oligonucleotide drug targeting the ACSL4 gene, and the operation steps are as follows: specifically binding to the ACSL4 mRNA sequence, forming a DNA-RNA hybrid chain with the ACSL4 mRNA, and using endogenous RNaseH1 to cut the RNA, thereby reducing the level of ACSL4 mRNA and protein; the antisense oligonucleotides of ACSL4 provided by the present invention include the following: ASO-1, ASO-2, ASO-3, ASO-4, ASO-5, ASO-6, ASO-7, ASO-8, ASO-9, ASO-10;

[0009] The sequence of ASO-1 is shown in SEQ ID No. 1: 5'-TGTACTCACCGGAACAGCAG-3';

[0010] The sequence of ASO-2 is shown in SEQ ID No. 2: 5′-GTACTCACCGGAACAGCAGC-3′;

[0011] The sequence of ASO-3 is shown in SEQ ID No. 3: 5′-TGGGCTTCAGTACAGTACAG-3′;

[0012] The sequence of ASO-4 is shown in SEQ ID No. 4: 5′-TTCAGTACAGTACAGTCTCC-3′;

[0013] The sequence of ASO-5 is shown in SEQ ID No. 5: 5′-TACTCACCGGAACAGCAGCC-3′;

[0014] The sequence of ASO-6 is shown in SEQ ID No. 6: 5′-TCCCAGCACCACATGATTCT-3′;

[0015] The sequence of ASO-7 is shown in SEQ ID No. 7: 5′-GCAGACATTCATGAATCGGT-3′;

[0016] The sequence of ASO-8 is shown in SEQ ID No. 8: 5′-CCAGCACCACATGATTCTGT-3′;

[0017] The sequence of ASO-9 is shown in SEQ ID No. 9: 5′-CAGACATTCATGAATCGGTG-3′;

[0018] The sequence of ASO-10 is shown in SEQ ID No. 10: 5'-CATTCATGAATCGGTGTGTC-3'.

[0019] Preferably, the sequence of the antisense oligonucleotide is modified with phosphorothioate (PS, indicated by *) throughout the chain, and 2'-O-methoxyethyl modification (2'-MOE) is introduced into the ribose of 5 bases at both ends, and the cytosine is all modified with methylcytosine. The sequence is any one of SEQ ID NO.1-SEQ ID NO.10 or any combination thereof, as shown in the following table:

[0020]

[0021] Preferably, the gene fragment is a sequence of SEQ ID No.3 or SEQ ID No.7;

[0022] The SEQ ID NO.3 sequence and modifications are as follows:

[0023] (MOE-T)*(MOE-G)*(MOE-G)*(MOE-G)*(MOE-5-Me-C)*(dT)*(dT)*(m5dC)*(dA)*(dG)* (dT)*(dA)*(m5dC)*(dA)*(dG)*(MOE-T)*(MOE-A)*(MOE-5-Me-C)*(MOE-A)*(MOE-G);

[0024] The SEQ ID NO.7 sequence is as follows:

[0025] (MOE-G)*(MOE-5-Me-C)*(MOE-A)*(MOE-G)*(MOE-A)*(m5dC)*(dA)*(dT)*(dT)*(m5dC )*(dA)*(dT)*(dG)*(dA)*(dA)*(MOE-T)*(MOE-5-Me-C)*(MOE-G)*(MOE-G)*(MOE-T);

[0026] Antisense oligonucleotides (ASOs) are artificially synthesized, chemically modified single-stranded oligonucleotides whose sequences can bind to target RNA bases in a complementary manner. After binding to RNA, ASOs will affect the activity of RNA in a variety of ways. The most common mechanism of action is that after binding to the target RNA, it will induce endogenous RNase H1 to specifically cleave the target RNA. The released ASO can continue to act on other target RNAs, thereby inhibiting the target gene. In order to improve the stability of ASOs in the body, chemical structure modification is also required to prevent rapid degradation in the body. ASO drugs have the advantages of high specificity, diverse modes of action, wide applicability, low immunogenicity, low toxicity and adverse reactions, and short research and development cycle, and have become the preferred means of treating various diseases.

[0027] Preferably, the antisense oligonucleotide sequence is fully phosphorothioate modified (PS, indicated by *), and 2'-O-methoxyethyl modified (2'-MOE) is introduced into the ribose of 5 bases at both ends, and the cytosine is all modified with methylcytosine.

[0028] Preferably, the antisense oligonucleotide sequence is subjected to other modifications, and the modification types include LNA, tricyclic DNA, 2'-O methyl, 2' cycloethyl (cET), UNA, 2' fluorine and conformationally restricted nucleosides.

[0029] The present invention designs 10 antisense oligonucleotides targeting the full length of ACSL4 according to the predicted RNA structure of ACSL4. The antisense oligonucleotide sequence provided by the present invention has obvious specificity and effect of inhibiting ACSL4.

[0030] The modified ACSL4 antisense oligonucleotides of the present invention mainly include the original antisense oligonucleotide sequences targeting ACSL4, and some special chemical modifications are added to make these sequences more stable and suitable for cell transfection experiments and commercial applications; the chemical modifications of the 10 antisense oligonucleotide sequences of the present invention mainly include the following specific modification types:

[0031] The phosphorothioate bond modification described in the present invention refers to the replacement of one oxygen atom in the phosphodiester bond (PO) with a sulfur atom (PS) in each nucleotide of the ASO molecule. This modification enhances the stability of the ASO against nucleases and enhances the affinity of the ASO for binding to proteins.

[0032] The 2'-O-methoxyethyl group modification described in the present invention refers to the 5 consecutive nucleotides at the 5'-end and 3'-end of the ASO molecule containing modified 2'-O-methoxyethyl (MOE) groups; the 2'-O-methoxyethyl group refers to the 3-methoxy modification (hydroxyl modified to methoxyethyl) at the 2' position of the furanose ring. The 2'-O-methoxyethyl group modification can enhance nuclease resistance, reduce off-target effects and enhance the hybridization affinity of antisense oligonucleotides.

[0033] The 5-methyldeoxyribocytidine (5-Me-dC) modification of the present invention refers to modifying the methyl group at the fifth position of the cytosine deoxyribonucleotide base; 5-Me-dC is complementary to the dG base, and this modification improves the stability of the double chain and can significantly reduce the immune response;

[0034] The present invention provides the results of Q-PCR and Western Blot after ASO transfection in cells, which show that ASO-3 and ASO-7 can effectively knock down the expression of ACSL4 protein, and the knockdown effect of ASO-7 is better, and 200nM can knock down more than 80%; the present invention also provides animal model experimental results, which show that knocking down ACSL4 in the rat brain can reduce the cerebral infarction area of ​​rats with ischemia-reperfusion injury.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The cell experiments and animal model experiments provided by the present invention prove that ACSL4 antisense oligonucleotides can effectively alleviate the damage to nerve cells during stroke. At the same time, the antisense oligonucleotides of the present invention, as a new type of nucleic acid drug, can be used for the auxiliary treatment of stroke and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the distribution diagram of the ASO of the present invention on the ACSL41 gene;

[0038] Figure 2 This is a graph showing changes in ACSL4 mRNA levels detected by Q-PCR 24 hours after ASO transfection in HEK293T cells of the present invention;

[0039] Figure 3 This is a graph showing changes in ACSL4 mRNA levels detected by Q-PCR 36 hours after transfection of ASO-3, ASO-7, and ASO-10 in HEK293T cells of the present invention;

[0040] Figure 4 This is a graph showing changes in ACSL4 protein levels detected by Western Blot 36 hours after transfection of ASO-3 and ASO-7 in HEK293T cells of the present invention;

[0041] Figure 5 It is a schematic diagram showing that transfection of ASO-7 in HEK293T cells of the present invention can reduce the MDA content caused by ferroptosis;

[0042] Figure 6 The neurobiological scoring of the present invention shows that ACSL4-ASO7 can attenuate the behavioral changes of neural damage caused by ferroptosis;

[0043] Figure 7 This is a diagram of TCC staining results after ACSL4-AS07 injection of the present invention;

[0044] Figure 8 This is a graph showing the ratio of rat cerebral infarction volume to total volume after TCC staining;

[0045] Fig. 9 This is a HE staining result diagram of the rat brain after ACSL4-AS07 injection in the present invention;

[0046] Fig.10 This is a statistical diagram of the volume percentage of rat cerebral ischemia-reperfusion injury after HE staining in the present invention. DETAILED DESCRIPTION

[0047] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0048] As attached Figure 1 To Attachment Fig.10 As shown:

[0049] Example 1: ASO transfection in HEK293T cells and detection of Acsl4 mRNA, the specific steps are as follows:

[0050] 1. Cell culture: HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum, and PC12 cells were cultured in MEM medium containing 10% fetal bovine serum (MEM is 1640 medium). Cells were inoculated 24 hours before transfection, and 2.5×10 cells were inoculated in each well of a 12-well cell culture plate. 4 When the cells grow to 70%-80% density, the next step of transfection is performed;

[0051] 2. Cell transfection: Take a corresponding amount of ASO and add it to 50 μL Opti-MEM, mix it thoroughly to make liquid A, take an appropriate amount of Celopener transfection reagent and add it to 50 μL Opti-MEM, mix it thoroughly to make liquid B, add liquid A drop by drop to liquid B, mix it thoroughly, let it stand at room temperature for 30 minutes, then add the mixed solution drop by drop to the well plate inoculated with cells, and culture the cells in a CO2 incubator at 37°C; the ASO is an antisense oligonucleotide, and ASO is used to downregulate the expression of the target gene by binding to mRNA and promoting its degradation;

[0052] 3. 8 hours after transfection, remove the cell culture medium containing the transfection reagent and add 1 mL of complete culture medium to each well;

[0053] 4. 24 or 36 hours after transfection, remove the cell culture medium, add 500 μL pre-cooled PBS buffer to each well, wash away the remaining culture medium;

[0054] 5. Add 500 μL trizol to each well to lyse the cells. After the cells are fully lysed, transfer the lysate to a 1.5 mL nuclease-free EP tube, add 100 μL chloroform, and shake thoroughly to mix;

[0055] 6. After standing at room temperature for 3 minutes, centrifuge at 12000g, 4 degrees Celsius for 15 minutes;

[0056] 7. After centrifugation, transfer 200 μL of the upper layer liquid to a new 1.5 mL nuclease-free EP tube, add an equal volume of isopropanol, shake thoroughly and mix, and centrifuge at 12000g, 4 degrees Celsius for 15 minutes;

[0057] 8. Keep the precipitate, remove the upper liquid, and rinse twice with 75% ethanol;

[0058] 9. After rinsing, remove 75% ethanol completely, leaving only the precipitate, and dry at room temperature for 5 minutes;

[0059] 10. Add 20 μL of nuclease-free water to dissolve RNA and measure RNA concentration using nanodrop2000;

[0060] 11. Take 1 μg for reverse transcription, the total reaction system is 20 μL, after the reverse transcription reaction is completed, add 80 μL of nuclease-free water to dilute the cDNA;

[0061] 12. Prepare the following mixture in a PCR tube

[0062]

[0063] The primers for human GAPDH were used as internal reference for qPCR, the upstream primer sequence was shown in Seq ID No.11: 5′-GGAGCGAGATCCCTCCAAAAT-3′, the downstream primer sequence was shown in Seq ID No.12: 5′-GGCTGTTGTCATACTTCTCATGG-3′, the primers for detecting gene ACSL4, the upstream primer sequence was shown in Seq ID No.13: 5′-CATCCCTGGAGCAGATACTCT-3′, the upstream primer sequence was shown in Seq ID No.14: 5′-TCACTTAGGATTTCCCTGGTCC-3′;

[0064] Depend on Figure 2 and Figure 3 As shown: Figure 2 Shows the ACSL4 mRNA level in HEK293T cells 24 hours after transfection with 100nM ACSL4 ASO; ASO-3 and ASO-7 have the best knockdown effect, with 100nM knockdown effect exceeding 50%;

[0065] Figure 3 It showed that the knockdown effects of ASO-3 and ASO-7 could reach 70% and 80% respectively after 36 hours of transfection at 200nM.

[0066] Example 2: HEK293T cell culture and ASO transfection and ACSL4 protein detection, the steps are as follows:

[0067] 1. Cell culture medium transfection: the steps are the same as above;

[0068] 2. Cell Harvest and Lysis

[0069] Cell harvesting: 24 or 36 hours after transfection, cells were collected by cell scraper.

[0070] Lyse cells: Use 200ul lysis buffer (add protease inhibitors before use at a ratio of 1:100) to lyse cells;

[0071] 3. Protein concentration determination: Use the BCA protein quantification method to determine the concentration of the protein sample; adjust the protein loading amount according to the measured protein concentration;

[0072] 4. Sample preparation: Add SDS sample buffer to the cell lysate, mix, and heat at 95°C for 5 to 10 minutes to denature the protein; load the sample into SDS-PAGE gel and perform electrophoresis with electrophoresis buffer;

[0073] 5. Transfer: transfer the electrophoretically separated proteins from the gel to polyvinylidene fluoride (PVDF);

[0074] 6. Blocking: Treat the membrane with blocking buffer (5% skim milk powder dissolved in PBST) at room temperature for 1 hour to reduce nonspecific binding;

[0075] 7. Primary antibody incubation: Incubate the membrane with a specific primary antibody (usually diluted in PBST containing a blocking agent), ACSL4 is diluted 1:1000, GAPDH is diluted 1:5000;

[0076] 8. Washing: Wash the membrane with PBST for 5 minutes each time, 3 times in total;

[0077] 9. Secondary antibody incubation: Incubate the membrane with secondary antibody containing HRP, goat anti-rabbit: 1:10000 dilution, goat anti-mouse: 1:10000 dilution; also include a washing step to remove excess secondary antibody;

[0078] 10. Detection: Use ECL (enhanced chemiluminescence) as a substrate to detect proteins.

[0079] Test results such as Figure 4 As shown, ASO-3 and ASO-7 can significantly reduce the expression of ACSL4 protein after transfection at 200 nM for 36 hours.

[0080] Example 3: Intrathecal injection (intrathecal (IT) injection is a method of delivering drugs or compounds directly into the cerebrospinal fluid of the spinal cord, and is often used to study the potential effects of drugs on the central nervous system). The following are the specific steps for intrathecal injection of ASO into rats:

[0081] 1. Preparation

[0082] ASO and syringe preparation: dilute ASO to 1.5 ug / uL as needed and load into a sterile syringe; Animal preparation: select healthy rats and group them according to the experimental design; ensure that the animals are in an empty stomach before the experiment to reduce the risk of vomiting during anesthesia;

[0083] 2. Anesthesia and disinfection

[0084] Anesthesia: Rats were given general anesthesia using a suitable anesthetic agent via intraperitoneal injection (e.g., isoflurane, propofol, etc.);

[0085] Disinfection: Fix the rat on the operating table, shave the hair at the injection site, and clean the skin with 70% alcohol or other disinfectants;

[0086] 3. Positioning and injection

[0087] 1) After the rat is anesthetized, shave the hair at the injection site and disinfect the skin surface at the injection site. Use the thumb and middle finger of the left hand to touch the iliac crests on both sides of the rat and tighten the skin on both sides. Use the index finger to touch the rat spine at the same level of the line connecting the iliac crests on both sides. The highest point touched is the L6 spinous process. Use a sterile microsyringe to vertically insert the needle into the gap between the L5 and L6 spinous processes. When a sense of emptiness appears, the rat's tail or hind limbs twitch slightly, and blood returns after a slight pull, it proves that the syringe has entered the subarachnoid space.

[0088] 2) Slowly tilt the syringe from vertical to 45°, and inject ASO once according to the weight of the rat. After the injection, retain the syringe for a period of time, slowly remove the microsyringe, and gently press the injection site for 1-2 minutes;

[0089] Note: It takes several attempts to find the right entry point. Once the needle enters the spine and there is no resistance when the needle is pushed forward, and a reflexive bounce of the tail or hind legs occurs, the needle has been correctly inserted.

[0090] Note: It is better to control the injection time within 10-30 seconds. Too fast speed will cause cramps in the rat's hind legs, which will disappear within 60 seconds.

[0091] After injection, the needle was rotated 180° 1-2 times and then removed from the spine;

[0092] The rats were returned to their cages for observation to confirm recovery of normal motor function.

[0093] Example 4: MDA content detection, the steps are as follows:

[0094] 1. Cell culture, transfection and harvesting: The steps are as described above;

[0095] 2. MDA determination: Detect the cell MDA content according to the instructions of Nanjing Jiancheng kit (A003-4-1). Transfer the cells to a centrifuge tube, add 0.5 ml of the extract, mix for 2 minutes, ultrasonically disrupt the cells to make a suspension, and take out the cells in each well after constant temperature oscillation at 37°C in a water bath for 1.5 hours;

[0096] 3. Add 2.0 mL of 10% trichloroacetic acid and 1.0 mL of 0.67% TBA, mix well, poke a small hole in the lid of each test tube, and react in a metal bath (95°C) for 15 min.

[0097] 4. Cool the sample with running water, centrifuge at 3500 r / min for 15 min, take the supernatant and compare the absorbance at 532 nm using an enzyme marker. The A532 value is used to represent the MDA level. The calculation formula is: MDA content ( nmol / mgprot) = (measured OD value - blank OD value) / (standard OD value - blank OD value) × standard concentration (10nmol / ml).

[0098] The results are as follows Figure 5 As shown: Cell transfection with ASO-7 can reduce the MDA content caused by ferroptosis.

[0099] Example 5: Reproduction of rat cerebral ischemia-reperfusion injury (MCAO / R) model

[0100] The rat middle cerebral artery occlusion / reperfusion (MCAO / R) model is a commonly used animal model for studying cerebral ischemia and reperfusion injury; this model simulates the process of vascular occlusion and subsequent blood flow restoration during human stroke, and its steps are as follows:

[0101] Preoperative preparation

[0102] 1) Anesthetize the rat: Anesthetize the rat using a suitable anesthetic (e.g., isoflurane or pentobarbital);

[0103] 2) Body temperature maintenance: During the surgery, a heating pad or lamp was used to maintain the rat’s body temperature;

[0104] Surgical procedures

[0105] 3) Incision: Make an incision of about 1.5 cm at 0.5 cm to the right of the middle of the rat's neck, ligate the proximal end of the CCA, clamp the distal end of the CCA with a venous clamp, tie a slipknot on the CCA with sutures, and cut a "V"-shaped notch 1 cm away from the bifurcation with ophthalmic scissors;

[0106] 4) Isolate blood vessels: Use silk thread to carefully isolate the blood vessels, taking care to avoid damaging the nerves;

[0107] 5) Insertion of the suture: Choose an embolization line (such as nylon line or silicone coated nylon line), insert the suture through the notch, loosen the artery clamp on the CCA, gently pull the ligature on the proximal end of the CCA to the right, adjust the angle of the line to the left to allow the suture to successfully enter the ICA, adjust the angle of the line to the right again by about 15°, and gently pull the CCA to allow the suture to enter the brain; cause local cerebral ischemia, and leave 1 cm of suture outside the incision; if it becomes difficult to enter after about 12 mm of insertion, it indicates that the PPA may have entered. At this time, withdraw the suture for a distance and adjust the angle of the line to the upper right again; if it is still not inserted correctly after repeated attempts, it means that the ICA has caused spasm, and blood retention can eliminate its spasm. At this time, withdraw the suture first, wait for blood flow to recover for 3-5 minutes, and then insert the suture again, which has a high success rate;

[0108] 6) Confirm MCAO: The thread plug is inserted to a depth of 18-20 mm (starting from the bifurcation of ECA and ICA). Stop when slight resistance is encountered (at this time, the right side of the rat's face will generally be observed to twitch). The tip of the nylon thread passes through the starting point of the MCA and reaches the thinner anterior cerebral artery. At this time, one side is completed.

[0109] Reperfusion

[0110] 7) Maintaining ischemia: The embolization line was placed for 120 minutes to simulate cerebral ischemia;

[0111] 8) Removal of the embolization line: At a set time point after ischemia, slowly and gently pull the nylon line back into the common carotid artery to achieve reperfusion of the middle cerebral artery;

[0112] 9) Surgical wound suture: Carefully suture the neck surgical incision after reperfusion, and keep the room temperature at 25℃-30℃ during ischemia and reperfusion;

[0113] Postoperative Management

[0114] 10) Postoperative care: After the rats regain consciousness, they are placed in cages and given fresh food and water. The animals’ body temperature, consciousness, and motor ability are monitored, and possible postoperative complications are promptly addressed.

[0115] 11) Medication: Antibiotics and analgesics will be given as appropriate.

[0116] After modeling, the Longa modified 5-point method was used for neurological scoring (see Table 1)

[0117] Table 1 Longa 5-point method

[0118]

[0119] Figure 6The results showed that after intrathecal injection of ACSL4-ASO (300 μg / rat) for 14 days, the MCAO (middle cerebral artery occlusion) animal model was established and ACSL4-ASO could significantly improve the neurobehavior of rats;

[0120] Figure 6 The neurobiological score of the present invention shows that ACSL4-ASO7 can attenuate the behavioral changes of neural injury caused by ferroptosis; wherein, Sham: sham operation group, MCAO / R model group, ### represents the model group, ** represents the ASO-treated model group, the model group is compared with the sham operation group, P < 0.001; ** represents the ASO-treated model group is compared with the untreated model group, P < 0.01.

[0121] Example 6: TTC staining and determination of cerebral infarction volume

[0122] The rat cerebral ischemia-reperfusion injury (MCAO / R) model was constructed as described in Example 4. After the experiment, the rats were anesthetized with 10% chloral hydrate (0.3 ml / 100 g), and the brains were quickly decapitated and the olfactory bulbs were removed. After freezing in a -20°C refrigerator for 10 minutes, the brain slices were taken out for continuous coronal sectioning, a total of 5 slices (one slice was about 2 mm); then the brain slices were placed in a 0.2% 2,3,5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride, TTC) solution, and stained at a constant temperature of 37°C in the dark for 15 minutes, and then the brain slices were taken out and placed in a 4% paraformaldehyde solution for fixation overnight; finally, the brain slices were taken out and photographed with a digital camera to evaluate the cerebral infarction of the brain tissue.

[0123] The infarct area was calculated using the Motic Images Plus 2.0 microscopic image analysis system (pink represents undamaged normal tissue, while the infarct area is not stained due to mitochondrial damage and is white). Since edema affects the infarct volume, a formula was used to correct it; the specific formula is as follows:

[0124] Volume of cerebral infarction lesion (mm 3 ) = total infarct area × brain slice thickness (2 mm)

[0125] Edema-corrected cerebral infarct volume (mm 3 ) = infarct volume × contralateral hemisphere volume / ipsilateral hemisphere volume;

[0126] Infarction rate (%) = edema-corrected cerebral infarction volume / bilateral cerebral hemisphere volume × 100%;

[0127] From the above Figure 7 and Figure 8 ; Figure 7The figure is the TCC staining result after ACSL4-AS07 injection of the present invention. Among them, Sham: sham operation group MCAO / R model group, ### represents the model group. Figure 8 This is a diagram of the total volume ratio of cerebral infarction volume in rats after TCC staining in the present invention, where Sham: sham operation group, MCAO / R model group, ### represents the model group, ** represents the AS0-treated model group, ### represents the comparison between the model group and the sham operation group, P<0.001; ** represents the comparison between the AS0-treated model group and the untreated model group, P<0.05.

[0128] Example 7: HE staining to detect cerebral infarction, the steps are as follows:

[0129] 1. At each time point after surgery, rats were anesthetized with chloral hydrate (0.3 mL / 100 g), their heads were cut off, their brains were removed, and their olfactory bulbs were removed;

[0130] 2. After freezing for 10 minutes, take out and make continuous coronal sections, a total of 5 slices (one slice is about 2 mm); take the 2nd, 3rd and 4th slices of brain tissue and fix them with 4% paraformaldehyde overnight, put them in a tissue dehydration box, rinse them with running water for 2 hours, and then put them in a tissue dehydrator for dehydration (see Table 2);

[0131] Table 2 Brain tissue dehydration steps

[0132]

[0133] After dehydration, the samples were embedded in a paraffin embedding machine, sliced ​​after cooling, with a thickness of 5 μm, and dried at 37°C for later use; the paraffin slices were placed in an oven for 30 min, and the slices were taken out of the oven for HE staining (see Table 3), sealed with neutral gum, and photographed;

[0134] Table 3 HE staining steps

[0135]

[0136] Finally get Fig. 9 and Fig.10 , Fig. 9 The figure is the HE staining result of the rat brain after ACSL4-AS07 injection in the present invention; wherein, Sham: sham operation group, MCAO / R model group; Fig.10 The volume percentage of rat cerebral ischemia-reperfusion injury was counted after HE staining in the present invention. Among them, Sham: sham operation group, MCAO / R model group, ### indicates model group, ** indicates AS0 treatment model group, ### indicates that the model group is compared with the sham operation group, P<0.001: ** indicates that the AS0 treatment model group is compared with the untreated model group, P<0.05.

[0137] The materials involved in the above content:

[0138] 1. Human embryonic kidney cells (human HEK293T cells); rats, SD, male, SPF grade, manufacturer: Sibeifu (Beijing) Biotechnology Co., Ltd.; suture: Beijing Xinong Technology Co., Ltd. Model: 2636A2;

[0139] 2. Serum-free culture medium for human cells: DMEM culture medium (Company: HyClone, Catalog Number: 11965126);

[0140] 3. Opti-MEM culture medium (Company: Thermo Fisher Scientific, Catalog Number: 11058021), Celopener transfection reagent (Company: Shanghai Jiying, Catalog Number: 13778150);

[0141] 4.TRIZOL (Company: Invitrogen (Shanghai) Catalog No.: 15596026); Hieff® qPCR SYBR® Green Master Mix (Company: Yisheng Biotechnology Catalog No.: 11203ES08) First Strand cDNA Synthesis Kit (Company: Thermo Fisher Scientific Catalog No.: K1622);

[0142] 5. Antibodies: Anti-ACSL4 antibody (Company: abcam Catalog No.: 1002422-15); anti-beta-tubulin mouse antibody (Company: Beijing Kangwei Century Biotechnology Co., Ltd. Catalog No.: 01270 / 07223).

[0143] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antisense oligonucleotide for inhibiting ACSL4 gene expression, characterized in that: The oligonucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.7.

Images