Application of miR-6340 in preparation of medicine for treating cerebral ischemia

Abstract

The invention discloses application of miR-6340 in preparation of a medicine for treating cerebral ischemia. Metabolism of GM1 in MCAO / R rats and metabolism of ganglioside GM1 in microglial cells after OGD / R can be inhibited by down-regulating expression of Glb1. It is found that upstream miRNA (miR-6340) of a Glb1 protein coding gene and mir-6340 / Glb1 promote the autophagy process of microglial cells through metabolism of GM1. According to the application, a new view angle is provided for the neuroprotective effect of GM1 on OGD / R induction by reducing autophagy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of miR-6340 in the preparation of drugs for treating cerebral ischemia. Background Art

[0002] Stroke is a common and refractory emergency disease, which is a group of diseases caused by sudden rupture of blood vessels in the brain or blockage of blood vessels leading to inability of blood to flow into the brain, resulting in brain tissue damage. Stroke is the leading common cause of death in humans and is also the main cause of disability, seriously endangering the quality of life and health of the people in our country. The per capita hospitalization cost of stroke patients increased by 31.4% in 2010 compared with that in 2005, bringing a heavy economic burden to the country and society. Stroke includes ischemic and hemorrhagic strokes, among which ischemic stroke, also known as ischemic stroke, accounts for 69.6% of strokes.

[0003] Recombinant tissue plasminogen activator (rt-PA) is currently the most commonly used intravenous thrombolytic drug for treating acute ischemic stroke. It can activate plasminogen and convert it into plasmin, and plasmin can degrade fibrin in thrombus, thereby dissolving thrombus and restoring cerebral blood perfusion. However, the narrow treatment window and side effects limit its clinical application. For example, symptomatic intracranial hemorrhage that may occur after thrombolysis can lead to aggravation of the patient's condition and even endanger life. According to statistics, the incidence of symptomatic intracranial hemorrhage after thrombolysis with rt-PA is about 6%.

[0004] Aspirin and clopidogrel are commonly used antiplatelet drugs, and their main problem is the risk of bleeding. Long-term use may lead to gastrointestinal bleeding. In particular, aspirin has a direct irritating effect on gastric mucosa and may cause gastric ulcer and gastric bleeding.

[0005] Edaravone is a free radical scavenger that can reduce the damage of free radicals generated during cerebral ischemia-reperfusion injury to nerve cells. However, during the use process, it may cause renal damage to elderly patients and patients with renal insufficiency, and its clinical efficacy is not very significant in some large-scale clinical trials.

[0006] Butylphthalide is a commonly used drug for improving cerebral circulation. It can increase cerebral blood flow in the ischemic area and improve microcirculation in patients with acute ischemic stroke. However, its optimal use time window is within 48 hours after onset, and the narrow treatment window problem also limits its clinical application. In addition, some patients may experience adverse reactions such as mild elevation of transaminase, nausea, and abdominal discomfort.

[0007] In addition to the above drug treatment methods, there are also treatment means such as endovascular intervention and angioplasty. Although they can quickly open occluded blood vessels and improve cerebral blood perfusion, their surgical operations are difficult and there are surgical operation risks. In addition, postoperative carotid artery restenosis is also a relatively common problem. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide an application of miR-6340 in the preparation of drugs for treating cerebral ischemia, which is specifically realized through the following technical solutions:

[0009] The first aspect of the present invention provides an application of miR-6340 in the preparation of drugs for treating cerebral ischemia.

[0010] Further, the treatment is achieved by reducing the area of cerebral infarction.

[0011] Further, reducing the area of cerebral infarction is achieved by down-regulating the expression of Glb1 to inhibit the metabolism of GM1.

[0012] Further, the treatment is achieved by reducing cell damage and autophagy.

[0013] Further, the autophagy is that miR-6340 / Glb1 regulates the autophagy process of BV2 cells through the metabolism of GM1.

[0014] Further, reducing cell damage is achieved by down-regulating the expression of Glb1 to inhibit the metabolism of GM1 in microglia after OGD / R.

[0015] The second aspect of the present invention provides a drug for treating cerebral ischemia, which contains miR-6340.

[0016] Glycosphingolipid biosynthesis-ganglioseries is a key metabolic pathway of stroke, and Glb1 is a key target on this metabolic pathway. This application locks the key target Glb1 for functional verification. The Glb1 gene encodes β-galactosidase. Inside the cell, β-galactosidase is located in an organelle called lysosome, which can decompose GM1 ganglioside and keratan sulfate.

[0017] This application establishes a rat cerebral ischemia-reperfusion model (MCAO / R) and an in vitro OGD / R model of microglia (BV2), microinjects Glb1-siRNA (in vivo siRNA) into the right cerebral ventricle to inhibit the target gene, and transfects Glb1-siRNA into BV2 cells in vitro experiments, to study and analyze whether Glb1 participates in the molecular mechanism of BV2 cell lesions during cerebral ischemia by regulating the synthesis of gangliosides. Predict and verify the miRNA (miR-6340) of the Glb1 protein-coding gene, and study the autophagy process of miR-6340 / Glb1 regulating BV2 cells through the metabolism of GM1.

[0018] In the brain atlas section of the HPA (Human ProteinAtlas) database of this application, the RNA expression levels of the Glb1 gene in the brain regions of three different mammals, namely humans, pigs, and mice, are shown. HE staining and double immunofluorescence staining experiments on brain slices of MCAO / R rats confirm the expression of the target gene Glb1 in brain cells on the ischemic side of the rats. After microinjecting Glb1-siRNA into the right cerebral ventricle of the rats, RT-qPCR and ELISA experiments show that downregulating the expression of Glb1 can inhibit the metabolism of GM1 in MCAO / R rats, thereby reducing the infarct volume.

[0019] After establishing an in vitro OGD / R model of microglia in this application, microglia are transfected with siRNA-Glb1. RT-qPCR, WB, Hoechst staining, immunofluorescence experiments, and ELISA experiments show that downregulating the expression of Glb1 can inhibit the metabolism of GM1 in microglia after OGD / R and reduce cell damage.

[0020] This application further predicts the miRNA (miR-6340) of the Glb1 protein-coding gene and confirms it through a luciferase reporter gene assay. Molecular dynamics simulation experiments show that the miR-6340—Glb1 ligand complex has high stability. RT-qPCR and WB experiments show that mir-6340 negatively regulates the expression of Glb1 in microglia. ELISA experiments show that miR-6340 / Glb1 is involved in the synthesis and metabolism of GM1 ganglioside in BV2 cells. RT-qPCR detects the mRNA levels of autophagy markers in BV2 cells after inhibiting the expression of Glb1. Transmission electron microscopy of cells further reveals that mir-6340 / Glb1 regulates the autophagy process of BV2 cells through the metabolism of GM1.

[0021] Explanation of the drawings in the specification

[0022] Figure 1 It is a schematic diagram of a stereotactic experiment on the brain;

[0023] Figure 2 It is the RNA expression level of the Glb1 gene in the human brain region;

[0024] Figure 3 It is the RNA expression level of the Glb1 gene in the brain regions of two different mammals, pigs and mice;

[0025] Figure 4 It is the pathological detection of the Glb1 protein in human tissues (A. Immunohistochemistry of the Glb1 protein in the human cerebral cortex. B. Results of double immunofluorescence staining of the Glb1 protein and human glioma cells);

[0026] Figure 5 It is the representative histopathological changes of brain slices in the rat MCAO / R model (100X);

[0027] Figure 6 It was for the immunofluorescence co-localization experiment after paraffin sectioning;

[0028] Figure 7 It was for detecting the mRNA level of Glb1 in the rat brain tissue after the MCAO / R model by RT-qPCR (Note: n = 6. ***P < 0.001, ****P < 0.0001);

[0029] Figure 8 It was for the representative MRI images of the rat MCAO / R model after microinjecting Glb1-siRNA and NC-siRNA (Note: Compared with the NC-siRNA group, **P < 0.01, n = 3, using t-test);

[0030] Figure 9 It was for detecting the expression of GM1 in the ischemic side brain tissue of rats by ELISA experiment (Note: *P < 0.05, ***P < 0.001, n = 6);

[0031] Figure 10 It was for detecting the expression of Glb1, p62, and LC3Ⅱ / Ⅰ in the ischemic side brain tissue of rats by WB experiment (Note: *P < 0.05, ***P < 0.001, n = 3);

[0032] Figure 11 It was for detecting the viability of BV2 cells after the OGD / R model (Note: (A) Cell phenotype changes at different reoxygenation times after 3 h of hypoxia; (B) Effects of different reoxygenation times on the survival rate of hypoxic damaged BV2 cells were measured by CCK-8 method, n = 6);

[0033] Figure 12 It was for detecting the expression of Iba1 by immunofluorescence (Note: Cells were treated with OGD for 3 h and then subjected to immunofluorescence experiment after 12 h of reoxygenation);

[0034] Figure 13 It was for detecting the effect of OGD / R on the expression of Glb1 in BV2 cells by RT-qPCR (Note: Cells were treated with OGD for 3 h and reoxygenated for 0 h, 6 h, 12 h, and 24 h. n = 3, **P < 0.01, compared with the normal group);

[0035] Figure 14 It was for detecting the effect of OGD / R on the expression of Glb1 in BV2 cells by WB (Note: Cells were treated with OGD for 3 h and reoxygenated for 0 h, 6 h, 12 h, and 24 h; n = 4, **P < 0.01, compared with the normal group);

[0036] Figure 15For Hoechst staining to detect cell apoptosis (Note: *P<0.05, **P<0.01, ***P<0.001, compared with the normal group);

[0037] Figure 16 For detecting the transfection efficiency of the FAM-siRNA vector;

[0038] Figure 17 For evaluating the efficiency of Glb1 silencing by RT-qPCR (Note: **P<0.01, ***P<0.001, compared with the normal group);

[0039] Figure 18 For establishing the OGD / R model after transfection and detecting the expression of Glb1 by RT-qPCR (Note: Cells were treated with OGD for 3 h and then reoxygenated for 12 h; **P<0.01, compared with the normal group. P<0.001, compared with the model group);

[0040] Figure 19 For detecting the mRNA levels of related enzymes in the GM1 ganglioside synthesis and metabolism pathways by RT-qPCR (Note: (A) Schematic diagram of the synthesis and metabolism process of GM1; (B) mRNA levels of a series of enzymes in the GM1 synthesis and metabolism pathways; **P<0.01, ****P<0.0001, n=3);

[0041] Figure 20 For molecular dynamics simulation experiments (Note: A. Free energy landscape diagram of the mir-6340—Glb1 complex - 2D; B. Free energy landscape diagram of the mir-6340—Glb1 complex - 3D; C. Number of intermolecular hydrogen bonds formed in the mir-6340—Glb1 complex; D. RMSD diagram of mir-6340 and Glb1; RMSF diagram of the Glb1 protein);

[0042] Figure 21 For dual-luciferase reporter assays (Note: *P<0.05);

[0043] Figure 22 For detecting the effect of OGD / R on the expression of mir-6340 in BV2 cells by RT-qPCR (Note: Cells were treated with OGD for 3 h and then reoxygenated for 0 h, 6 h, 12 h, and 24 h; n=6, *P<0.05, **P<0.01 compared with the normal group);

[0044] Figure 23 For detecting the effect of transfection of miRNA inhibitor and miRNA mimics on the expression of Glb1 in BV2 cells by WB (Note: *P<0.05, ***P<0.001);

[0045] Figure 24To detect the synthesis of GM1 ganglioside by Elisa assay (Note: A. Cell culture supernatant sample; B. Cell extract sample; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0046] Figure 25 To detect the mRNA levels of autophagy markers by RT-qPCR (Note: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0047] Figure 26 The ultrastructure of BV2 cells under transmission electron microscope (15000x, 1μm) (Note: A: Control group; B: OGD / R group; C: OGD / R + mir-6340 inhibitor group; D: OGD / R + siRNA-NC group; E: OGD / R + Glb1-siRNA). Specific embodiments

[0048] The specific embodiments of the present invention will be described in detail below.

[0049] I. Experimental materials and methods

[0050] 1. Experimental animals: 30 male SD rats (SPF grade, body weight 300 ± 20 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd.; the rats were housed in the Experimental Animal Center of Zhejiang Chinese Medical University, and the experimental animal use license number was SYXK (Zhe) 2021-0012. The room temperature was 25 ± 1°C, the relative humidity was 50% - 60%, and the light-dark cycle was 12 h / 12 h. They could freely access food and water. All animals were strictly in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals, 2011). The animal experiment protocol and guidelines were approved by the Institutional Animal Care and Use Committee of the Experimental Animal Research Center of Zhejiang Chinese Medical University.

[0051] Experimental groups: ① Sham group (sham operation group), ② MCAO / R group (rat middle cerebral artery occlusion model group), ③ MCAO / R + NC-siRNA group (MCAO / R + right ventricle microinjection - empty control group), ④ MCAO / R + Glb1-siRNA group (MCAO / R + right ventricle microinjection of Glb1-siRNA).

[0052] 2. Experimental cells: The microglial cells used in this experiment were derived from the second-generation BV2 cells (purchased from the Cell Bank of Shanghai Institute of Life Sciences, Chinese Academy of Sciences).

[0053] Experimental grouping: ① Control group (normal group), ② OGD / R group (oxygen-glucose deprivation / reperfusion model group), ③ OGD / R + inhibitor-NC group (OGD / R + inhibitor-negative control group), ④ OGD / R + miRNA inhibitor group (OGD / R + mir-6340-inhibitor group), ⑤ OGD / R + NC-siRNA group (OGD / R + negative control-siRNA group), ⑥ OGD / R + Glb1-siRNA group, ⑦ OGD / R + miRNA inhibitor + Glb1-siRNA group.

[0054] 3. Experimental reagents

[0055] CRUZ

[0056]

[0057] ZOMANBIO

[0058]

[0059]

[0060] Camilo

[0061]

[0062] 4. Experimental equipment

[0063]

[0064] Company

[0065]

[0066] II. Experimental methods

[0067] 1. Establish a rat MCAO / R model: Select healthy adult SD rats weighing about 250 - 300 g. Fix the rats in the supine position on the operating table and secure the limbs and head with tape. Use an electric hair clipper to remove the hair in the middle of the rat's neck, ranging from the mandible to the suprasternal fossa. Disinfect the surgical area with povidone-iodine, cover with a sterile drape, and only expose the surgical field. Neck incision and blood vessel exposure: Make a longitudinal incision about 2 - 3 cm long in the middle of the neck. Use ophthalmic scissors and forceps to carefully separate the subcutaneous tissue and bluntly separate the muscle layer along both sides of the trachea to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). During the separation process, pay attention to avoiding damage to the surrounding nerves, such as the vagus nerve and sympathetic nerve, and minimize bleeding at the same time. Artery ligation and preparation for inserting the suture: First, ligate the distal end of the external carotid artery with a silk thread and make a slipknot (loose knot) at the proximal end. Then, use a microvascular clip to temporarily clamp the common carotid artery and internal carotid artery to block blood flow. Make a small incision at the proximal end of the ligation site of the external carotid artery, and the size of the incision should be just suitable for inserting the suture. Suture insertion and positioning: Slowly insert the pre-prepared suture from the incision of the external carotid artery, and then gently pass the suture through the bifurcation of the external carotid artery - common carotid artery and into the internal carotid artery. During the insertion process, keep the direction of the suture correct and the movement gentle. When the suture is inserted to a certain depth (generally about 18 - 20 mm, and the specific depth may vary slightly depending on the size of the rat and the vascular anatomical structure), a slight resistance will be felt. At this time, the tip of the suture usually reaches the origin of the middle cerebral artery, thus blocking the blood flow of the middle cerebral artery and causing local cerebral ischemia. After that, tighten the slipknot on the external carotid artery to fix the suture.

[0068] Ischemic period: Keep the suture inserted to maintain continuous ischemia of the middle cerebral artery. The ischemic time can be set according to the experimental purpose, generally 1 - 2 hours, to simulate different degrees of cerebral ischemic injury. During the ischemic period, closely observe the vital signs of the rats, such as respiration and heart rate, and at the same time pay attention to keeping the rat's body temperature stable. You can use a heating pad and other equipment to maintain the rat's body temperature at about 37°C.

[0069] Reperfusion period: After the predetermined ischemic time, carefully pull out the suture, release the vascular clips on the common carotid artery and internal carotid artery, restore blood flow, and start the reperfusion stage. After reperfusion, also closely observe the changes in the neurological function of the rats, such as limb movement and balance ability, as well as the pathophysiological changes in the brain tissue.

[0070] 2. HE staining

[0071] 2.1 Sample pretreatment steps

[0072] (1) Fixation of brain tissue with paraformaldehyde: Five days after the MCAO / R model, SD rats were anesthetized with Zotazone 50 (intramuscular injection, 40 mg / kg). After fully exposing the heart, a perfusion needle was inserted from the apex to the left ventricle, and the auricle was quickly cut open to form a perfusion fluid discharge channel. 100 mL of normal saline was quickly pushed into the syringe, and then 200 mL of 4% paraformaldehyde solution was slowly and evenly pushed into the syringe for fixation. After the perfusion was completed, the intact brain was quickly removed and placed in a 4% paraformaldehyde solution for fixation for 24 h.

[0073] (2) Brain tissue fixation: Cut brain tissue of appropriate size and place it in an embedding frame. Fix it in neutral buffered formalin (pH 7.4) for 2 h. Dehydrate the fixed brain tissue in an automatic dehydrator.

[0074] (3) Sample wax embedding: Place the transparent tissue block in the melted paraffin, put it in a wax melting box and keep it warm for 3 hours, and use 60-62℃ paraffin wax specially used for pathological sections to embed the tissue.

[0075] (4) Sample sectioning: Use a paraffin microtome to slice the sample to a thickness of 4 μm, pick up the slices, and bake them in a 60°C oven for 2 h.

[0076] 2.2 HE staining steps

[0077] Pretreated sections were dewaxed in xylene for 10 minutes twice, followed by dewaxing with graded ethanol solutions (100%, 95%, 80%, and 70%) until the surface was free of water. After hydration, sections were placed in hematoxylin stain for 10 minutes and then washed with distilled water for 2 minutes. The sections were then dehydrated in 95% ethanol for 3 minutes twice and stained with 0.5% alcohol eosin for 2 minutes. Sections were then dehydrated in 80% and 95% ethanol for 3 minutes each twice, and then dehydrated in 100% ethanol for 3 minutes twice. Sections were then cleared in xylene for 5 minutes twice. The sections were then mounted and observed under a light microscope for histopathological features and images were acquired.

[0078] 3. Immunofluorescence double staining experiment

[0079] 3.1 Sample pretreatment steps: Same as 2.1.

[0080] 3.2 Immunofluorescence detection steps

[0081] (1) Sample dewaxing and dehydration: Sample dewaxing and gradient rehydration: The pretreated samples were dewaxed in xylene for 10 min, twice, and then dewaxed in 100%, 95%, 80%, and 70% gradient ethanol solutions in sequence until they were naturally hydrated.

[0082] (2) Antigen retrieval: Place the sections in a citrate buffer solution with a concentration of 0.1 mol / L and a pH of 6. Heat them in a microwave oven at medium power for 2 minutes, then stop for 1 minute. Then heat them at low-medium power for 2 minutes. After stopping the heating, keep them covered for 15 minutes. Wash them with PBS solution for 5 minutes, and repeat this 3 times in total. Then place the sections in a 3% H2O2 aqueous solution for 10 minutes, and wash them with PBS solution for 5 minutes again, repeating this 3 times in total.

[0083] (3) Incubate the samples with primary antibodies: Use an immunohistochemistry pen to draw a circle (the pen should not touch water). Drop two different primary antibodies diluted in an appropriate ratio on the section samples, and add 0.3% tritonX-100. Incubate them overnight at 4°C in a wet box.

[0084] (4) Incubate the samples with fluorescent secondary antibodies: Wash them with PBS solution for 5 minutes, repeating this 3 times in total. Drop two different fluorescent secondary antibodies on the washed samples and incubate them at 37°C for 60 minutes. Then rinse them with PBS solution for 5 minutes, repeating this 3 times in total.

[0085] (5) DAPI staining: Mount the slides with an anti-fluorescence quencher containing DAPI.

[0086] (6) Microscopic observation: Place the processed samples under the microscope to observe the number of positive cells in the brain tissue sections.

[0087] 4. Intracerebroventricular injection of Glb1-siRNA (in vivo siRNA) into rats

[0088] Rats were fasted for 12 hours before surgery. Anesthetize SD rats with Zoletil 50 (intramuscular injection, 40 mg / kg), and use a hair clipper to remove the hair on the head. Make a midline incision on the rat's head and peel off the fascia until the intersection of the coronal suture and the sagittal suture (bregma) is completely exposed. According to the rat brain stereotaxic atlas, find the right cerebral ventricle of the rat, which is 1.5 mm lateral to the bregma point, 1.1 mm posterior to the bregma, and 4.5 mm deep (coordinates are ±1.5, -1.1, -4.5), and mark the injection point with a pencil. Randomly divide the rats into the NC-siRNA group and the Glb1-siRNA group. The NC-siRNA group is the negative empty control group. Inject slowly at a rate of 2 μL / min, with an injection dose of 10 μL, a total of 10 nmol. Leave the needle in place for 10 minutes after injection. After the surgery, disinfect and suture the scalp. Both groups of rats were prepared with the MCAO / R model by the suture occlusion method, and the other operations were the same. Figure 1 The flow chart of the brain stereotaxic experiment is shown.

[0089] 5. Magnetic resonance imaging (MRI) to detect the cerebral infarction volume in rats

[0090] After 5 days of modeling, the rats in each group were anesthetized with Zoletil 50. The rats lay prone on the scanning bed with their heads fixed. Magnetic resonance imaging (MRI) measurements were performed using a 7 Tesla 20 cm bore superconducting magnet (Magnex Scientific Inc) interfaced with a Bruker Advanced Console (Bruker Biospin MRI). A three-plane scout imaging sequence was used to adjust the position of the animal's head. The repetition time TR = 3500 ms, the echo time TE = 102 ms, the scan thickness was 2 mm, and the entire brain of the rat was scanned. Utilizing the inherent atomic nuclei in the animal body, resonance occurs under the action of an external magnetic field, absorbing energy and releasing MR signals. These signals were collected and used as the imaging source, and after computer processing, an MR image of the animal body was formed, and T2WI images of the cerebral infarction site were obtained. The Image J image analysis software was used to measure the volume of cerebral infarction in rats.

[0091] 6. RT-qPCR experiment

[0092] According to the instructions of the Cell / Tissue Total RNA Isolation Kit V2, the brain tissues of the rats in each group were placed on ice and ground and lysed thoroughly. After multiple washes to remove impurities, the total RNA of the samples was extracted. Using the 5×All-In-One RT MasterMix kit, the total RNA was reverse transcribed into cDNA through a 20 μL reverse transcription reaction system. The reaction program was: 25 °C, 10 min; 42 °C, 15 min; 85 °C, 5 min.

[0093] Using the Applied Biosystems QuantStudio 3 software system, the primer sequences are shown in Table 1. A 20 μL amplification system was used: 10 μL Universal SYBR Green Fast qPCR Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL cDNA, 7.2 μL ddH2O for real-time PCR analysis. The PCR thermal cycling steps were as follows: pre-denaturation: 95 °C, 3 min; cycle reaction: 95 °C, 5 s, 60 °C, 30 s for 40 cycles; melting curve: 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s. The PCR products were analyzed using the melting curve to confirm the correctness of the amplification. According to the obtained CT values, with GAPDH as the internal reference gene and the sham operation group as the control group, the relative mRNA expression levels of each gene were calculated. The calculation formula was:

[0094] Calculation formula:

[0095] Table 4-1 Primer sequence table

[0096]

[0097]

[0098] 7. Western Blot (WB)

[0099] (1) Gel preparation: Prepare 12.6% separating gel and 5% stacking gel successively according to the molecular weight of the target protein.

[0100] (2) SDS-PAGE electrophoresis: Prepare electrophoresis buffer. The sample loading amount is 10 - 20 μg, and an equal volume of the quantified protein sample is added to each well. Cover the electrophoresis tank lid and connect the power supply. The electrophoresis conditions are first 80 V for 30 min, and then 120 V for 80 min. Electrophoresis buffer: Weigh 1.0 g of SDS, 14.40 g of Glycine, and 3.00 g of Tris-base powder, add ddH2O and stir to dissolve it completely, and finally quantify to 1 L.

[0101] (3) Transfer: Prepare transfer buffer. The PVDF membrane is activated with methanol, and the protein is transferred to the PVDF membrane. The black side (negative electrode) is placed at the bottom layer, followed by sponge, filter paper, gel, PVDF membrane, and then filter paper and sponge. The white side (positive electrode) is placed on the top. Electrophoresis conditions: 0.3 A for 2 h. Place the electrophoresis tank in a container filled with ice for electrophoresis. 10× Transfer Buffer stock solution: Weigh 144.13 g of Glycine and 30.28 g of Tris-base powder, add ddH2O and stir to dissolve it completely, and finally quantify to 1 L; 1× Transfer Buffer: Measure 50 mL of 10× Transfer Buffer stock solution, add 100 mL of methanol and 350 mL of ddH2O, mix well and use.

[0102] (4) Blocking: Prepare 5% skim milk with 1×TBST and block the PVDF membrane on a shaker for 1 h. 1×TBST solution: Weigh 8.70 g of NaCl, add 250 mL of 2 M Tris-HCl and 1 mL of Tween 20, add ddH2O and stir to mix well, and finally quantify to 1 L.

[0103] (5) Primary antibody incubation: Dilute the primary antibody (1:1000) with antibody diluent according to the antibody instruction manual, and incubate the PVDF membrane with the primary antibody at 4°C overnight.

[0104] (6) Secondary antibody incubation: Quickly wash the PVDF membrane twice with 1×TBST and then wash it 3 more times, 10 min each time. Dilute the HRP-conjugated secondary antibody (1:5000) with 1×TBST solution and incubate it at room temperature for 1 h.

[0105] (7) Development: Wash the PVDF membrane three times with 1×TBST solution for 10 min each time. Gently add the developer mixture to the PVDF membrane and incubate at room temperature for 1 min. Then, transfer the PVDF membrane to a developer and perform quantitative analysis using ImageJ software.

[0106] 8. Detection of Ganglioside Expression by Enzyme-Linked Immunosorbent Assay (ELISA)

[0107] (1) Prepare standard samples: Let the freeze-dried rat GM1 standard stand for 5 minutes and then centrifuge slowly. Then add 1.0 mL of standard diluent and mix gently after it is fully dissolved (concentration is 20 ng / mL). Then mark ① on the tube. Take 7 centrifuge tubes and mark them separately.

[0108] ②, ③, ④, ⑤, ⑥, ⑦, ⑧. Add 300 μL of standard diluent to each tube. Remove 300 μL from tube ① and add it to tube ②. Mix thoroughly. Then remove 300 μL from tube ② and add it to tube ③. Repeat this process until tube ⑦. Tube ⑧ contains the standard diluent and serves as a blank control. The final concentrations in each tube are: 20, 10, 5, 2.5, 1.25, 0.625, and 0.312 ng / mL, respectively. This serves as the standard curve.

[0109] (2) Sample detection: 100 μL per well was added to the ELISA plate, with samples from each group or different concentrations of rat GM1 standard (0 ng / mL well plus standard dilution) added; incubated at 37°C for 90 min.

[0110] (3) Wash the plate twice with washing buffer. Add 100 μL of rat GM1 antigen working solution to each well (prepare the rat GM1 antigen working solution 30 minutes in advance). Incubate at 37°C for 60 minutes.

[0111] (4) Wash the plate three times. Add 100 μL of enzyme conjugate working solution to each well except the blank well. Prepare the solution 30 minutes in advance in the dark. Seal the wells with adhesive tape and incubate at 37°C in the dark for 30 minutes.

[0112] (5) Wash the plate 5 times with the washing solution. Add 100 μL of TMB colorimetric working solution to each well (including the blank well) and incubate at 37°C in the dark. Stop the incubation when the color of the high concentration of the standard curve is darker and there is a clear color gradient.

[0113] (6) Add 100 μL of stop solution to each well, including the blank well, mix well and measure immediately. Set the microplate reader to an OD value of 450 nm.

[0114] 9. Cell Culture

[0115] 9.1 Cell Recovery

[0116] (1) Prepare a 15 mL sterile centrifuge tube and add 8 mL of culture medium.

[0117] (2) Take out the cryopreservation tube from the liquid nitrogen container, immerse it in warm water at 37 °C, and shake it from time to time to make it melt quickly.

[0118] (3) Take a cotton ball containing 75% alcohol to wipe the laminar flow hood. Take out the cryopreservation tube from the 37 °C water bath, transfer the cells in the tube to the centrifuge tube containing 8 mL of culture medium prepared, and gently pipette to disperse the cells evenly.

[0119] (4) Centrifuge at 1,000 rpm for 5 min. Discard the supernatant, add 1 mL of fresh culture medium, and gently pipette to form a cell suspension.

[0120] (5) Count the cells, adjust the cell density, inoculate into a culture flask, aspirate the cell suspension into a T25 cell flask, supplement the culture medium to 5 mL, gently shake the cell flask to make the cells distribute evenly, and incubate statically in a 37 °C incubator.

[0121] (6) Observe the cell attachment and growth conditions, and change the culture medium once the next day.

[0122] 9.2 Cell passage

[0123] (1) Passage the cells when they reach 80% to 90% confluence. Aspirate the old culture medium in the flask and wash the cells 2 - 3 times with PBS solution.

[0124] (2) Add an appropriate amount of digestive solution (trypsin) to the flask and gently shake the culture flask to make the digestive solution flow over the surface of all cells.

[0125] (3) Incubate in the incubator for 50 s. When it is found that the cytoplasm retracts and the cell gap increases, immediately terminate the digestion and add 2 volumes of complete culture medium to terminate the digestion.

[0126] (4) Aspirate the culture medium in the flask, repeatedly pipette the cells on the flask wall to make the cells detach from the flask wall to form a cell suspension. Transfer the cells to a 15 mL centrifuge tube and centrifuge at 1,000 rpm for 5 min.

[0127] (5) Pour out and discard the supernatant, add pre - warmed complete culture medium to resuspend the cells, count, dilute the cell suspension to an appropriate cell seeding density, transfer it to a new cell culture flask, and place it in the cell culture incubator for culture.

[0128] 9.3 Cell cryopreservation

[0129] (1) When the cell confluence reaches 80% to 90%, perform cell cryopreservation. Follow the above passage steps, digest the monolayer - grown cells with trypsin and centrifuge to obtain a cell pellet.

[0130] (2) Add an appropriate amount of cryopreservation solution and gently pipette to make the cells uniform. After adjusting the cell concentration, transfer 1 mL of the cell suspension to a cryotube. Mark the name of the cells, the cryopreservation time, and the operator on the cryotube.

[0131] (3) Place the cryotube containing the cells in an -80 °C refrigerator overnight and finally transfer it to a liquid nitrogen tank.

[0132] 10. Glb1-siRNA transfection

[0133] (1) Seed BV2 cells into the culture wells of a 6-well plate containing an appropriate amount of complete medium at a density of 30×10 4 cells / well (v1). (2) Dilute siRNA: Dilute 5 μL of 20 μM siRNA stock solution (v3) with 120 μL of 1×riboFECT TM CP Buffer (v2) and mix gently. (3) Prepare the mixture: Add 12 μL of riboFECT TM CP Reagent (v4), gently pipette to mix, and incubate at room temperature for 15 min to prepare the transfection complex. (4) Add the riboFECT TM CP transfection complex to an appropriate amount of antibiotic-free complete medium (v1) and mix gently. (5) Incubate the culture plate in a 37 °C CO2 incubator for 24 h.

[0134] 11. Establishment of the cell OGD / R model

[0135] The cells are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in a 37 °C, 5% carbon dioxide incubator. The condition for establishing the OGD / R model is 3 h of hypoxia. When the cells reach 80% confluence in the culture dish by area, remove the medium and wash the cells with PBS. Then add serum-free and sugar-free DMEM to the cells, place them in a self-made hypoxia chamber, seal it, and culture for 3 h to make the cells hypoxic. The control group of BV2 cells is cultured in a normal incubator. Reoxygenate the BV2 cells with a normal 10% serum concentration medium for 0 h, 6 h, 12 h, and 24 h.

[0136] 12. Cell viability assay

[0137] Seed the cells into a 96-well plate. After OGD / R treatment, according to the manufacturer's instructions, use the Cell Counting Kit-8 (CCK-8) to detect and evaluate cell viability. Add 10 μL of CCK-8 solution to each well and incubate at 37 °C for 1 h. At the same time, measure the optical density (OD) value at a wavelength of 450 nm. Cell survival rate (%) = (OD value of OGD / R - OD value of blank well) / (OD value of normal control group - OD value of blank well) × 100%.

[0138] 13. Detection of cell apoptosis by Hoechst staining

[0139] The apoptosis of BV2 cells was detected using the DNA-binding dye Hoechst 33342. After treatment under OGD / R conditions, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature and incubated with 10 μg / mL Hoechst 33342 in the dark at 37 °C for 25 min. Then the cells were washed three times with PBS and immediately observed under a fluorescence microscope. Cells showing strong blue fluorescence and nuclear condensation were scored as apoptotic cells. The percentage of apoptotic cells was calculated by the ratio of apoptotic cells to the total number of counted cells.

[0140] 14. Dual-luciferase reporter assay

[0141] (1) BV2 cells were routinely cultured under the conditions of 37 °C and 5% CO2.

[0142] (2) BV2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 1.0×10 4 cells per well with a total volume of 100 μL per well and cultured in an incubator at 37 °C for 24 h.

[0143] (3) Dilute miRNA mimics or Non-target Control, the 3’UTR dual reporter gene vector or mutant vector of the target gene with 5 μL of serum-free medium, and dilute 0.25 μL of Lipo6000TM transfection reagent with 5 μL of medium. Let them stand for 5 min respectively, then mix them, shake gently, and let them stand for 5 min. The final concentration of mimics is 50 nM, and the plasmid dosage is 100 ng. Each group has 3 replicate wells. Replace the culture medium 6 h after transfection.

[0144] (4) After 48 h of transfection, aspirate the culture medium and add 300 μL of lysis buffer. Incubate on ice for 5 min to lyse the cells.

[0145] (5) Take 20 μL of the lysed sample solution and add it to the culture plate. Set 3 replicate wells according to the experimental needs.

[0146] (6) Dilute the firefly luciferase substrate (50×) and the Renilla luciferase substrate (50×) with the corresponding buffer to 1× working solution. Prepare in advance, protect from light and incubate to room temperature.

[0147] (7) Add 100 μL of the firefly luciferase reaction solution, shake the plate to mix well, and detect the activity of firefly luciferase. The detection is completed within 30 min.

[0148] (8) Add 100 μL of Renilla luciferase reaction solution, mix well on a shaking plate, and measure the activity of Renilla luciferase. The measurement should be completed within 30 min. The ratio of the firefly fluorescence signal value to the Renilla fluorescence signal value is used as the final value for each group.

[0149] 15. Observation of autophagy in cells by transmission electron microscopy

[0150] (1) After digesting BV2 cells, neutralize them with medium or serum and transfer them into a 1.5 mL centrifuge tube. Centrifuge for 3 min at 1000 rpm. Gently aspirate the supernatant along the tube wall, add 500 - 1000 μL of 5% serum, resuspend the cells, and then centrifuge again.

[0151] (2) Gently aspirate the supernatant. Keep the sample in a lump all the time. Wash it once with 1 mL of PBS.

[0152] (3) Remove the supernatant, and then add 1 mL of 2.5% glutaraldehyde solution (the volume must be more than 20 times the volume of the cell mass. If it becomes turbid, it cannot be used). You can scrape along the tube wall with a toothpick to separate the cells from the tube wall (about the size of a millet grain), so that each cell mass can fully contact the fixative. Fix at 4 °C for more than 4 h.

[0153] (4) Fix with 2.5% glutaraldehyde, wash with PBS, re-fix with 1% osmium tetroxide, dehydrate step by step with acetone, soak in Epon-812 embedding agent, and embed and cure. Ultra-thin sections (70 nm) are double-stained with uranyl acetate and lead citrate. Observe the changes in autophagy structures in cells under a transmission electron microscope.

[0154] 16. Statistical analysis of data

[0155] Experimental data are expressed as mean ± standard deviation (SD). Statistical analysis is performed using GraphPad Prism 8.0.1 software. A t-test is used for significant analysis of comparisons between two groups. One-way analysis of variance (ANOVA) or non-parametric tests are used for analysis of comparisons among multiple groups. P < 0.05 indicates that the data are statistically significant.

[0156] III. Experimental results

[0157] (I) Effects of the common target Glb1 of three types of prescriptions on microglia

[0158] 1. HPA database

[0159] To study the expression levels of Glb1 protein in human tissues and cells, this part of the study utilized the BrainAtlas and pathological section data in the HumanProteinAtlas database (https: / / www.proteinatlas.org / ). The HPA database is a free public database containing more than 26,000 antibodies targeting more than 17,000 human genes. The BrainAtlas shows the RNA expression of different proteins in the mammalian brain by integrating three different mammalian species: human, pig, and mouse. RNA-seq results generated by the Genotype-Tissue Expression (GTEx) project indicate that the main expression sites of Glb1 in the human brain are the pituitary gland, retina, and cerebral cortex; in the mouse brain, the main expression sites are the pituitary gland, hypothalamus, and cerebral cortex; and in the pig brain, the main expression sites are the spinal cord, pituitary gland, and midbrain ( Figure 2-3 ,, Note: Image source: HPA database (https: / / www.proteinatlas.org / ENSG00000170266-GLB1)). The pathological section results in human brain tissues show that Glb1 has a high expression in the cerebral cortex. Meanwhile, the immunofluorescence results in human glioma cells (U-251MG) show that Glb1 is expressed in all cells ( Figure 4 , Image source: HPA database (https: / / www.proteinatlas.org / ENSG00000170266-GLB1)).

[0160] 2. Histopathological changes in brain sections of the rat MCAO / R model

[0161] After the MCAO / R model, blood was taken from the abdominal aorta, and the brain was removed by perfusion with 4% paraformaldehyde. The abdominal aorta was clamped with an arterial clamp, a perfusion needle was inserted through the left ventricle and fixed, the right auricle was cut open, and 100 mL of normal saline was rapidly perfused first, followed by a uniform and slow perfusion of 200 mL of 4% paraformaldehyde. After the brain was removed, it was placed in 4% paraformaldehyde for later use. After the fixed brain sections underwent dehydration and embedding steps, serial paraffin sections were prepared, and sections with a thickness of 4 μm were taken for HE staining. HE staining showed that the cells in the normal group were intact and arranged neatly, while the brain tissue around the parietal cortex of MCAO / R rats was loose and edematous, the nuclei were vacuolated, and there were demyelination changes around ( Figure 5 ).

[0162] 3. Expression of common targets in brain cells on the ischemic side of rats

[0163] Double immunofluorescence labeling of Glb1 with endothelial cell, astrocyte, microglia, and neuron cell markers was used on paraffin sections of the brains of the rat MCAO / R model. The number and phenotypes of the labeled cells in the MCAO / R model group were compared. Figure 6The results showed that the co-localization of Iba1 and Glb1 was significantly higher than that of other markers, indicating that Glb1 was highly expressed in microglia after modeling. Therefore, microglia were selected for subsequent experiments.

[0164] (2) Downregulating the expression of Glb1 can inhibit the metabolism of GM1 in MCAO / R rats

[0165] 1. Expression of Glb1 after intracerebroventricular injection of Glb1-siRNA

[0166] After intracerebroventricular injection of Glb1-siRNA (in vivo siRNA) and NC-siRNA into the right cerebral ventricle of rats, in order to detect the effectiveness of siRNA transduction, RT-qPCR experiments were performed in this application to detect the expression of Glb1 in the right cerebral ventricle of rats. Compared with the Sham group, the mRNA level of Glb1 in the MCAO / R group increased significantly (P < 0.001); compared with the MCAO / R group, there was no significant change in the mRNA level of Glb1 in the MCAO / R + NC-siRNA group, indicating that the NC-siRNA empty control had no effect on the expression of Glb1 in rats; compared with the MCAO / R + NC-siRNA group, the mRNA level of Glb1 in the MCAO / R + Glb1-siRNA group decreased significantly (P < 0.0001), indicating that the inhibitory efficiency of in vivo injection of Glb1-siRNA was high and subsequent experiments could be carried out ( Figure 7 ).

[0167] 2. Inhibiting the expression of Glb1 can reduce the infarct area in MCAO / R rats

[0168] Glb1-siRNA and NC-siRNA were intracerebroventricularly injected into the right cerebral ventricle of rats, and then MCAO / R modeling was performed in both groups. After 5 days, magnetic resonance imaging (MRI) was used to evaluate the infarct area and detect the degree of brain injury. As Figure 8 shown, the infarct volume of rats microinjected with Glb1-siRNA decreased significantly (P < 0.01).

[0169] 3. Inhibiting the expression of Glb1 can inhibit the metabolism of GM1 in MCAO / R rats

[0170] Weigh 0.05 g of tissue, add 500 μL of PBS with a pH of 7.2, homogenize the tissue thoroughly with a homogenizer, centrifuge and collect the supernatant. The expression of GM1 in each group was detected by Elisa experiment, and the results are as Figure 9As shown, compared with the Sham group, the expression of GM1 in the MCAO / R group was significantly decreased (P<0.001); compared with the MCAO / R group, there was no significant change in the GM1 level in the MCAO / R+NC-siRNA group, indicating that the NC-siRNA negative control had no effect on the expression of GM1 in rats; compared with the MCAO / R+NC-siRNA group, the expression level of GM1 in the MCAO / R+Glb1-siRNA group was significantly increased (P<0.05), suggesting that inhibiting the expression of Glb1 could inhibit the metabolism of GM1 in MCAO / R rats.

[0171] 4. Inhibiting the expression of Glb1 can reduce the autophagy level in MCAO / R rats

[0172] Through literature review, it was found that oxidative stress culture showed an increase in senescence-related -β-galactosidase (Glb1 / SAβ-gal), leading to strong changes in the lysosomal system, including an increase in lysosomal mass and the content of autophagosomes, accumulation of oxidized substances and damaged mitochondria in lysosomes. Therefore, in this application, WB experiments were used to detect the expression of autophagy markers p62 and LC3Ⅱ / Ⅰ in the ischemic side brain tissue of rats. As Figure 10 shown, compared with the Sham group, the expression level of Glb1 in the MCAO group was significantly increased (P<0.05), the expression level of p62 was significantly decreased (P<0.05), and the expression level of LC3Ⅱ / Ⅰ was significantly increased (P<0.001). Compared with the MCAO group, there were no significant changes in the expression levels of Glb1, p62, and LC3Ⅱ / Ⅰ in the MCAO+NC-siRNA group; compared with the MCAO+NC-siRNA group, the expression level of Glb1 in the MCAO+NC-siRNA group was significantly decreased (P<0.05), the expression level of p62 was significantly increased (P<0.05), and the expression level of LC3Ⅱ / Ⅰ was significantly decreased (P<0.05).

[0173] (3) Downregulating the expression of Glb1 can inhibit the metabolism of GM1 in microglia after OGD / R

[0174] 1. Trend of cell phenotype change of BV2 cells with reoxygenation time after OGD / R model

[0175] Control group BV2 cells were cultured in a normal incubator. BV2 cells were reperfused with a normal 10% serum concentration medium, and the reoxygenation times were 0 h, 6 h, 12 h, and 24 h. The optimal reoxygenation time was screened by CCK8 experiment. The hypoxia time was 3 h, and the optimal reoxygenation time was 12 h for subsequent mechanism research ( Figure 11 ).

[0176] 2. BV2 cells were activated after OGD / R

[0177] Microglia were treated with OGD / R and reperfused for 0 h, 6 h, 12 h, and 24 h. After fixation with 4% paraformaldehyde at room temperature for 30 min; blocked with 1% BSA for 1 h, incubated with the primary antibody (Iba1 1:200) overnight at 4°C; the next day, after washing with PBS, labeled with the fluorescent secondary antibody (1:1000) for 1 h, stained with DAPI for 5 min; after mounting, scanned with a fluorescence microscope. The immunofluorescence results further confirmed that after 12 h of reoxygenation with OGD / R, the expression of the activation marker Iba1 of microglia was significantly increased, indicating that microglia were activated( Figure 12 ).

[0178] 3. Expression level of Glb1 in BV2 cells after OGD / R model

[0179] The total RNA of BV2 cells was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, real-time fluorescence quantitative PCR was performed using 2X Universal SYBR Green Fast qPCR Mix. The experiment was repeated 3 times. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal reference index for the control experiment, and the 2-ΔΔCt method was used to analyze its relative expression level. The results of real-time fluorescence quantitative PCR showed( Figure 13 ), compared with the control group, the mRNA expression level of Glb1 increased with the reoxygenation time at 0 h, 6 h, and 12 h after OGD / R in microglia (P<0.01). The results of Western-Blot are shown in Figure 14 , compared with the normal group, the protein expression level of Glb1 was significantly increased after OGD / R-6h and OGD / R-12h (P<0.01).

[0180] 4. Detection of cell apoptosis after OGD / R model by Hoechst staining

[0181] An OGD / R model was established, and cell apoptosis was detected by Hoechst staining. As Figure 15 shown, the genomic DNA of cells in the normal group was evenly distributed, and it was a relatively uniform blue after Hoechst staining, and the cell nucleus was round. In apoptotic cells, due to nuclear condensation, deformation and fragmentation, the nuclear shape of Hoechst staining became crescent-shaped or fragmented into several pieces, and the blue color presented was white and bright. Compared with the normal group, the cell proliferation decreased and the apoptotic cells increased significantly after OGD / R-12h (P<0.001).

[0182] 5. Transfection of Glb1-siRNA into microglia

[0183] 5.1 Transfection efficiency

[0184] To detect the transfection efficiency of siRNA, the green light of FAM-siRNA was observed under a fluorescence microscope and compared with the field of view under a light microscope (the number of cells was counted under white light and fluorescence respectively). As Figure 16 shown, the time point with the highest siRNA transfection efficiency was 24 h after transfection. The concentration of siRNA was 100 nM, and the transfection efficiency of the cells had reached over 80%, so the subsequent experiments could be carried out.

[0185] 5.2 Expression of Glb1 in BV2 cells after transfection with Glb1-siRNA

[0186] In this experiment, three kinds of siRNAs targeting Glb1 and negative control NC-siRNA were synthesized, and the final transfection concentration of siRNA was 100 nM. The efficiency of Glb1 silencing was determined by RT-qPCR, and the Glb1-siRNA with the highest silencing efficiency was selected for subsequent mechanism research. BV2 cells were divided into 5 groups: (1) normal group, (2) normal group + NC-siRNA, (3) Glb1-siRNA-1, (4) Glb1-siRNA-2, (5) Glb1-siRNA-3. The expression of Glb1 after transfection was detected by RT-qPCR. As Figure 17 shown, we observed that Glb1-siRNA-3 showed the best interference efficiency (P < 0.001), and the treated cells were labeled as the Glb1-siRNA group.

[0187] An OGD / R model was established 24 h after transfection, and the expression of Glb1 was detected by RT-qPCR. BV2 cells were divided into 5 groups: (1) Control, (2) Control + NC-siRNA, (3) Model group, (4) Model + NC-siRNA group, (5) Model + Glb1-siRNA group. As Figure 18 shown, compared with the normal group, the mRNA level of Glb1 in the model group was significantly increased (P < 0.01); compared with the model group, there was no obvious change in the mRNA level of Glb1 in Model + NC-siRNA, indicating that the empty vector control NC-siRNA had no effect on the expression of Glb1 in BV2 cells; compared with the Model + NC-siRNA group, the mRNA level of Glb1 in the Model + Glb1-siRNA group was significantly decreased (P < 0.001), indicating that the inhibitory efficiency of in vivo injection of Glb1-siRNA was relatively high, and the subsequent experiments could be carried out.

[0188] 6. Downregulation of Glb1 expression can inhibit the metabolism of GM1 in BV2 cells

[0189] The OGD / R model was established 24 h after Glb1-siRNA transfection. The mRNA levels of a series of enzymes in the GM1 ganglioside synthesis and metabolism pathways were measured by RT-qPCR, including the biosynthetic synthases GM3, GM2, and GM1, and the mRNA expression levels of Glb1, Hexa, Neu1, GALC, and GBA during GM1 metabolism. Figure 19 The results showed that compared with the normal group, the expression levels of the GM1 biosynthetic synthases GM3, GM2, and GM1 in the OGD / R group were significantly decreased, indicating a weakened GM1 synthesis pathway; meanwhile, there was an upward trend in the expression levels of GM3, GM2, and GM1 after Glb1-siRNA transfection. Compared with the normal group, the expressions of Glb1 and Hexa in the GM1 metabolic pathway in the OGD / R group were increased, and the mRNA expression levels of Neu1, GALC, and GBA were decreased, indicating an enhanced early and weakened late GM1 metabolic pathway; meanwhile, after Glb1-siRNA transfection, the expression levels of Glb1 and Hexa were weakened, and the mRNA expression levels of Neu1, GALC, and GBA all showed a downward trend, indicating a weakened GM1 metabolic process.

[0190] (IV) mir-6340 / Glb1 is involved in the metabolism of GM1 ganglioside in BV2 cells

[0191] 1. Prediction of miRNA targeting the gene Glb1 and molecular dynamics simulation of mir-6340—Glb1

[0192] To clarify the molecular mechanism by which the target gene regulates the activation of BV2 cells in ischemic stroke, miRNAs were screened through three databases, TargetScan, MiRDB, and miRWalk. The results of software analysis showed that there was a high possibility of binding between the 3'-untranslated region of Glb1 mRNA and mmu-miR-6340, and there was a binding site.

[0193] To further verify the interaction between Glb1 and mir-6340, this application conducts molecular dynamics simulation experiments. Based on the basic principles of Newtonian mechanics and using computer software, it simulates the interaction and movement change laws of macromolecules. The free energy landscape (Gibbs Energy Landscape) is obtained by trajectory sampling starting from different polypeptide conformations. It can show the valleys and hills of free energy experienced by the polypeptide during the entire simulation process. Blue indicates lower energy, and red indicates higher energy. RMSD is the root mean square deviation of atomic positions, which refers to the average distance measured between atoms of two different conformation proteins. The change trend of the RMSD of proteins and ligands is also an important characterization for judging whether the simulation reaches stability. RMSF refers to a method of measuring the deviation of atomic position changes over time relative to the reference position, which can characterize the flexibility and movement intensity of protein amino acids during the entire simulation process. Hbonds refers to a hydrogen bond analysis tool. ​ C Calculated the number of hydrogen bonds in the ligand-protein complex. Hydrogen bonds between amino acids can change the internal geometry of the protein. ​ D Indicated that the RMSD value of the Glb1 protein was low, indicating high stability of the protein during the simulation time. At the same time, the small molecule mir-6340 ligand remained flat after experiencing fluctuations. Usually, amino acids close to the active site would show lower RMSF values. ​ E The results showed that the RMSF value of the Glb1 protein was low, indicating a higher ligand-protein interaction during the simulation time. In summary, the molecular dynamics simulation experiment further verified the stable and reliable binding between mir-6340 and Glb1.

[0194] 2. Dual-luciferase reporter gene

[0195] The dual-luciferase reporter gene assay verifies the interaction between miRNA and the target gene by the downregulation of the relative fluorescence value of the reporter gene. The pmirGLO dual-luciferase reporter vector is used, and the reporter fluorescence of the vector is the Renilla luciferase gene (hRluc), and the corrected fluorescence is the firefly luciferase gene (hluc). The 3'UTR region of the gene is cloned downstream of the hRluc gene. miRNA acts on the target gene through the 3'UTR region, so miRNA is co-transfected with the constructed reporter gene vector. The ratio of the firefly fluorescence signal value to the Renilla fluorescence signal value is used as the final value for each group.

[0196] As ​The results showed that the luciferase reporter gene assay indicated that after normalization and correction with the NC group, the reporter fluorescence of Glb1-MUT pmirGLO increased and recovered compared with that of Glb1-WT pmirGLO after co-transfection with mir-6340 mimic (note *P < 0.05). These results suggested that mir-6340 interacted with the Glb1 3'UTR locus.

[0197] 3. OGD / R treatment induced downregulation of mir-6340 expression in BV2 cells

[0198] Reverse transcription used the stem-loop method for miRNA cDNA and detected the miR-6340 level. The primer sequences and stem-loop sequences were listed in Table 2. The RT-qPCR results showed that compared with the control group, the miR-6340 level was significantly downregulated in BV2 cells treated with OGD / R for 0 h, 6 h, 12 h, and 24 h (P < 0.05), and reached a lower level at 6 h and 12 h after reperfusion ( ​ ).

[0199] Table 2: Primer sequences of miR-6340

[0200]

[0201]

[0202] 4. mir-6340 regulated Glb1 expression

[0203] Chemical methods were used to synthesize and construct miRNA inhibitor and miRNA mimics to inhibit and enhance the regulatory effects of mir-6340, respectively. The cell suspensions containing mir-6340 inhibitor / inhibitor-NC and mir-6340 mimics / mimics-NC were transferred to 6-well cell culture plates, respectively, at a transfection concentration of 100 nM. After culturing for 48 h, the cells were collected for subsequent Western blotting experiments to detect the expression of the target gene Glb1. The Western blotting experiments showed ( ​ ), compared with the inhibitor-NC group, the inhibitor mir-6340 inhibitor group upregulated the expression of Glb1 in microglia (P < 0.001); compared with the mimics-NC group, the overexpression mir-6340 mimics group downregulated the expression of Glb1 in microglia (P < 0.05). The results suggested that mir-6340 negatively regulated the expression of Glb1 in BV2 cells.

[0204] 5. mir-6340 / Glb1 was involved in the synthesis and metabolism of GM1 ganglioside in BV2 cells

[0205] Microglia were co-transfected with Glb1-siRNA and mir-6340 inhibitor. After plating and culturing microglia for 24 h, the culture medium was changed and mir-6340 inhibitor and Glb1-siRNA were transfected. After culturing for 24 h, the cells were subjected to OGD / R treatment. The culture medium of each group of cells was taken, centrifuged for 20 min, and the supernatant was carefully collected. According to the specified procedure in the kit instructions, the relative expression of GM1 ganglioside in the supernatant of the culture medium of each group of cells was detected by ELISA experiment. The collected cells of each group were washed 3 times with cold PBS. 200 μL of lysis solution was added to every 1×106 cells to break the cells. The extract was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken for detection.

[0206] The ELISA results of the cell culture supernatant samples and cell lysate samples both showed ( ​ ), compared with the normal group, the expression of GM1 was down-regulated in the OGD / R model group (P < 0.0001); compared with the control group intervened with inhibito-NC, the expression of GM1 in microglia was down-regulated in the OGD / R model group intervened with inhibito-NC (P < 0.01); compared with the OGD / R model group intervened with inhibito-NC, the expression of GM1 in microglia was down-regulated in the OGD / R model group intervened with mir-6340 inhibitor (P < 0.001); compared with the OGD / R model group intervened with inhibito-NC, the expression of GM1 in microglia was down-regulated in the OGD / R model group intervened with mir-6340 inhibitor and siRNA-NC (P < 0.05); compared with the OGD / R model group co-intervened with mir-6340 inhibitor and siRNA-NC, the expression level of GM1 in microglia was up-regulated in the OGD / R model group co-intervened with mir-6340 inhibitor and Glb1-siRNA (P < 0.0001), indicating that OGD / R induced the synthesis of GM1 ganglioside in microglia through the mir-634- / Glb1 axis. In addition, the experimental results showed that compared with the cell lysate samples, the concentration of GM1 was higher in the cell supernatant culture medium and the data changes of each group were more significant. According to relevant literature reports, GM1 is an acidic glycosphingolipid that forms lipid rafts in the outer leaflet of the cytoplasmic membrane, especially in neurons of the central nervous system. They are involved in cell proliferation, differentiation, adhesion, signal transduction, cell-cell interaction, and tumorigenesis. Therefore, this application further confirmed that the concentration of GM1 decreased in BV2 cells after OGD / R, and thus the signal transduction and interaction between cells may also decrease accordingly, which requires further experimental verification. <00>

[0207] (5) mir-6340 / Glb1 promotes the autophagy process of BV2 cells through the metabolism of GM1

[0208] 1. Detection of the mRNA levels of autophagy markers in BV2 cells after downregulating Glb1 expression by RT-qPCR

[0209] Previous studies have found that oxidative stress culture shows an increase in senescence-associated β-galactosidase (SA-Glb1 / SAβ-gal), leading to strong changes in the lysosomal system, including an increase in lysosomal mass and the content of autophagic vacuoles. At the same time, the GM1 metabolic pathway is closely related to cell autophagy. Therefore, in this application, the mRNA levels of autophagy markers in BV2 cells after downregulating Glb1 expression were detected by RT-qPCR. The RT-qPCR results showed ( ​ ), compared with the Control group, after OGD / R treatment of BV2 cells, the mRNA levels of ATG7, Bcn1, LC3A, and LC3B were all increased and statistically significant. There was no significant difference in the mRNA levels of Lamp1 and Lamp2, but there was still an upward trend, indicating that the autophagy level of cells increased after OGD / R treatment. Compared with the Model group, the mRNA levels of ATG7, LC3A, and LC3B in the Model+Glb1-siRNA group were significantly decreased and statistically significant, indicating that the mRNA levels of autophagy markers in BV2 cells decreased after inhibiting Glb1 expression, and the autophagy level of cells decreased.

[0210] 2. Observation of autophagy in BV2 cells after transfection with Glb1-siRNA by transmission electron microscopy

[0211] Observation by transmission electron microscopy found that all specimens in each group showed good observability under the microscope, with good staining of the cell nucleus and cytoplasm and uniform coloring, indicating that the fixation, sectioning, and specimen preparation met the standards. Further observation found that compared with the cells in the normal group, the cell nucleus of the specimens in the OGD / R group was significantly enlarged, and there were autophagosome structures with double membranes ( ​ B). A large number of vacuoles wrapped by single membranes were visible in the OGD / R+mir-6340 inhibitor group and the OGD / R+siRNA-NC group, and the cytoplasmic components had been degraded ( ​ C, D). Compared with the OGD / R group, autophagosomes were occasionally seen in the cells of the OGD / R+Glb1-siRNA group ( ​E), and the degree of mitochondrial damage is reduced. After treatment with the OGD / R + mir-6340 inhibitor group, the expression of Glb1 in cells will increase, while after treatment with the OGD / R + Glb1-siRNA group, the expression of Glb1 in cells will decrease. The experimental results show that after OGD / R, the expression of Glb1 in cells increases, and at the same time, the autophagy level in cells also increases. Therefore, we have reason to infer that after BV2 cells are treated with OGD / R, mir-6340 / Glb1 causes cell damage by participating in the autophagy process of BV2 cells.

Claims

1. Use of miR-6340 in the preparation of a drug for treating cerebral ischemia.

2. The application according to claim 1, characterized in that, The treatment is achieved by reducing the cerebral infarction area.

3. The application according to claim 2, wherein Reducing the cerebral infarction area is achieved by down-regulating the expression of Glb1 to inhibit the metabolism of GM1.

4. The application according to claim 1, characterized in that, The treatment is achieved by reducing cell damage and autophagy.

5. The application according to claim 3, wherein The autophagy is that miR-6340 / Glb1 regulates the autophagy process of BV2 cells through the metabolism of GM1.

6. The application according to claim 3, characterized in that, Reducing cell damage is achieved by down-regulating the expression of Glb1 to inhibit the metabolism of GM1 in microglia after OGD / R.

7. A drug for treating cerebral ischemia, characterized in that, The drug contains miR-6340.

Images