WO2018153244A1 - 己糖激酶2特异性抑制剂在急性中枢神经系统损伤疾病中的应用 - Google Patents

己糖激酶2特异性抑制剂在急性中枢神经系统损伤疾病中的应用 Download PDF

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WO2018153244A1
WO2018153244A1 PCT/CN2018/075401 CN2018075401W WO2018153244A1 WO 2018153244 A1 WO2018153244 A1 WO 2018153244A1 CN 2018075401 W CN2018075401 W CN 2018075401W WO 2018153244 A1 WO2018153244 A1 WO 2018153244A1
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hexokinase
antibody
hypoxia
microglia
brain
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PCT/CN2018/075401
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English (en)
French (fr)
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颜光美
银巍
李媛
陆秉政
盛龙祥
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中山大学
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Priority to IL268753A priority Critical patent/IL268753B2/en
Priority to BR112019017661-6A priority patent/BR112019017661A2/pt
Priority to US16/488,011 priority patent/US11584802B2/en
Priority to KR1020197027839A priority patent/KR102359994B1/ko
Priority to NZ756855A priority patent/NZ756855A/en
Priority to SG11201907581VA priority patent/SG11201907581VA/en
Priority to JP2019546304A priority patent/JP6932197B2/ja
Priority to CA3053866A priority patent/CA3053866C/en
Application filed by 中山大学 filed Critical 中山大学
Priority to AU2018223394A priority patent/AU2018223394B2/en
Priority to RU2019128326A priority patent/RU2736499C1/ru
Priority to EP18757439.7A priority patent/EP3586873A4/en
Publication of WO2018153244A1 publication Critical patent/WO2018153244A1/zh
Priority to AU2020202576A priority patent/AU2020202576B2/en
Priority to US18/095,660 priority patent/US20230167195A1/en

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    • A—HUMAN NECESSITIES
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    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions

  • the invention belongs to the field of biomedicine and relates to the application of a specific inhibitor of hexokinase 2 in the prevention and treatment of acute central nervous system injury diseases.
  • Stroke also known as stroke, is an acute cerebrovascular disease, a disease caused by sudden rupture of blood vessels in the brain or cerebral blood flow caused by vascular occlusion, including ischemic and hemorrhagic stroke. Among them, ischemic stroke accounted for about 85% of the total cases.
  • t-PA Current tissue plasminogen activator
  • the immune system-mediated inflammatory response following acute ischemic stroke is a widely studied therapeutic target.
  • the results of clinical trials of drugs using this mechanism as therapeutic targets are not satisfactory.
  • Fingolimod and Natalizumab which are agents of the peripheral immune system inflammatory response, are effective in inhibiting the penetration of lymphocytes into the brain parenchyma, but clinical trials have shown that receiving treatment does not Stroke patient groups benefit. Therefore, in-depth exploration of the central nervous system inflammatory response mediated by microglia after ischemia is expected to provide new therapeutic targets and strategies for the prevention and treatment of acute ischemic stroke.
  • the inventors of the present invention obtained a series of glycolytic pathway genes by screening, and identified that the selective up-regulation of hexokinase 2 mediates the activation process of hypoxia-induced microglia.
  • the inventors have confirmed that a series of biologically active substances having a selective inhibitory activity of hexokinase 2 can inhibit hypoxia-induced activation of microglia.
  • the inventors have also found that hexokinase 1 and hexokinase 3 interference cannot inhibit hypoxia-induced activation of microglia.
  • the present invention provides the use of a hexokinase 2 specific inhibitor for the preparation of a medicament for the prevention and treatment of an acute central nervous system injury disorder.
  • Another aspect of the invention provides the use of a composition comprising a specific inhibitor of hexokinase 2 for the manufacture of a medicament for the prevention and treatment of acute central nervous system damaging diseases.
  • a further aspect of the invention provides a method of preventing and treating an acute central nervous system injury disease, the method comprising administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a hexokinase 2 specific inhibitor or comprising a hexose A composition of a kinase 2 specific inhibitor.
  • the present inventors have discovered a neuroprotective effect of a hexokinase 2 specific inhibitor in the prevention and treatment of acute central nervous system injury.
  • Cytological experiments and in vivo animal experiments indicate that up-regulated expression of selective hexokinase 2 regulates hypoxia-induced activation of microglia and microglia-mediated neuroinflammatory responses after ischemia, whereas hexokinase 2 selection
  • Both sex inhibitors and gene knockdown significantly inhibit microglia-mediated inflammatory responses and thereby exert neuroprotective effects.
  • Figure 1 Enhancement of the glycolytic pathway is essential for the activation of microglia by hypoxia.
  • E–F hypoxia had no effect on the survival rate of BV 2 cells.
  • (I) Graphical representation of key metabolites in the glycolytic pathway; markers of markedly enhanced metabolites (n 4) after exposure to hypoxia for 6 h.
  • (K) Decreased percentage of activated cells after treatment with 2-DG in (J). The scale is 50 ⁇ m (n 4).
  • FIG. 2 Hypoxia-induced microglial activation involves up-regulation of hexokinase family members.
  • FIG. 3 Hexokinase 2 interference is effective in inhibiting the activation of microglia induced by hypoxia.
  • BV 2 cells were transfected with or without HK2 siRNA for 24 h and stimulated for an additional 24 h under hypoxic conditions, and the levels of the indicated proteins were analyzed by Western blotting.
  • B BV2 cells transfected into different interference fragments of HK2 can effectively inhibit the morphological changes of microglia induced by hypoxia.
  • C The percentage decrease in activated glial cells of HK2 knockdown cells under hypoxic conditions.
  • D HK2 knockdown significantly inhibited the expression of CD11b.
  • FIG. 4 Hexokinase 1 and hexokinase 3 interference do not inhibit hypoxia-induced activation of microglia.
  • Using the HK1 and HK3 interfering fragments to interfere with hexokinase 1 and hexokinase 3, respectively (A and C), did not effectively inhibit the morphological changes associated with activation of microglia caused by hypoxia (B and D) (n 3).
  • FIG. 5 Pyruvate kinase M2 subtype (PKM2) interference does not inhibit hypoxia-induced activation of microglia.
  • PKM2 Pyruvate kinase M2 subtype
  • FIG. 6 The hexokinase 2 inhibitor lonidamine is effective in inhibiting the activation of microglia induced by hypoxia.
  • A Activation status of pMG and BV 2 cultures during hypoxia was significantly inhibited in the presence of lonidamine (50 ⁇ M). The scale is 50 ⁇ m.
  • B The picture shows a decrease in the percentage of microglia activated after treatment with lonidamine in (A).
  • C Immunofluorescence assay showed that BV 2 and pMG cells under hypoxia stimulation had decreased expression of CD11b in the presence of lonidamine. The scale is 50 ⁇ m. * ⁇ 0.05; ** ⁇ 0.01; *** ⁇ 0.001.
  • Figure 7 Up-regulated expression of hexokinase 2 results in an increase in histone acetylation and transcriptional expression of the pro-inflammatory cytokine Il1b.
  • B (D) HK2 inhibition reverses the accumulation of intracellular acetyl-CoA in BV 2 cells and inhibits the up-regulation of acetylated histones.
  • FIG. 8 Lonidabamine effectively protects rats from brain damage caused by middle cerebral artery infarction.
  • A Representative pictures of 2,3,5-triphenyltetrazolium chloride (TTC) stained brain sections in each treatment group.
  • Figure 9 In vivo gene knockdown of hexokinase 2 effectively protects rats from brain damage caused by middle cerebral artery infarction.
  • FBP fructose-1,6-diphosphate
  • G-3-P glyceraldehyde 3-phosphate
  • IL-1 ⁇ interleukin-1 ⁇
  • LPS lipopolysaccharide
  • rAAV recombinant adeno-associated virus
  • ROS active oxygen
  • siRNA short interfering RNA
  • TTC 2,3,5-triphenyltetrazolium chloride.
  • the invention provides the use of a hexokinase 2 specific inhibitor for the manufacture of a medicament for the prevention and treatment of an acute central nervous system injury disorder.
  • the hexokinase 2 specific inhibitor refers to a substance capable of specifically or selectively inhibiting the biological activity of hexokinase 2 (also referred to as hexokinase II or HK2).
  • the hexokinase 2 specific inhibitor comprises an antibody to hexokinase 2 or a fragment thereof.
  • the antibody refers to a protein capable of specifically binding to hexokinase 2 and inhibiting or quenching the activity of hexokinase 2.
  • Fragments of antibodies can include, for example, Fab, Fab', (Fab') 2, and Fv. The production and purification of antibodies or fragments thereof are known in the art.
  • a hexokinase 2 antibody of the invention may also exist in the form of an amino acid sequence, a nucleotide sequence, an expression vector comprising the nucleotide sequence or amino acid sequence encoding the antibody.
  • the hexokinase 2 antibodies of the invention may also be present in an expression vector or host cell in the form of a fusion protein.
  • the hexokinase 2 specific inhibitor comprises a substance capable of specifically inhibiting translation of mRNA of hexokinase 2, or a substance capable of specifically degrading mRNA of hexokinase 2, such as siRNA, shRNA, The miRNA or its modifications, thereby interfering with the synthesis of hexokinase 2 by the RNAi mechanism.
  • siRNA, shRNA or miRNA can be obtained by in vitro synthesis techniques, which are well known in the art.
  • the siRNA, shRNA or miRNA of the invention is present in a particular vector, such as a cell.
  • the acute central nervous system injury disease refers to a disease or condition of central nervous system damage caused by acute ischemia or hypoxia, including but not limited to, acute spinal injury, brain trauma, retinal damage, lack of Oxygen brain injury, acute ischemic brain injury, ischemic stroke, hypoxic stroke, neonatal hypoxic ischemic encephalopathy, toxic encephalopathy, acute cerebral infarction, lacunar infarction, transient brain Brain and spinal cord nerve damage caused by ischemic attack, severe craniocerebral injury, cerebrospinal surgery and brain spinal cord radiotherapy.
  • the invention provides the use of a composition comprising a specific inhibitor of hexokinase 2 for the manufacture of a medicament for the prevention and treatment of an acute central nervous system injury disorder.
  • the hexokinase 2 specific inhibitors included in the composition are as described above, and the composition may further comprise other hexokinase 2 inhibitors.
  • the other hexokinase 2 inhibitors include, but are not limited to, 2-deoxyglucose; Lonidamine; bromopyruvic acid; glucose 6-phosphate; Imatinib; 5-thio-glucose; methyl jasmonate.
  • a further aspect of the invention provides a method of preventing and treating an acute central nervous system injury disease, the method comprising administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a hexokinase 2 specific inhibitor or comprising a hexose A composition of a kinase 2 specific inhibitor.
  • the subject refers to a mammalian subject, such as a human.
  • the administration means administration of a drug to, for example, a human body by subcutaneous, transdermal, intramuscular, intravenous, intraarterial, sublingual, buccal, gastrointestinal, or the like.
  • a hexokinase 2 specific inhibitor eg, a nucleic acid form
  • a further aspect of the present invention provides a method for preventing and treating an acute central nervous system injury disease, which comprises selectively or specifically reducing or inactivating the activity of hexokinase 2 in a subject in need thereof.
  • the activity of reducing or inactivating hexokinase can be achieved, for example, by genetic engineering means to reduce or eliminate the expression of the hexokinase 2 protein.
  • An exemplary means is to alter the sequence of the hexokinase 2 encoding nucleotide by site directed mutagenesis.
  • Another exemplary means is the translation process that interferes with the mRNA of hexokinase 2 by RNAi technology. Any method known to those skilled in the art to reduce the expression of a particular protein in a cell is contemplated for use in the methods of the invention.
  • mouse BV2 microglia cell line high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), 2-deoxyglucose (Sigma-Aldrich, D8375), 2-( N-7-nitro-2,1,3-benzothiazol-4-amino)-2-deoxyglucose (2-NBDG, Thermo Fisher Scientific, N13195), Annexin/PI staining kit (Biotool, B32115) , Flow Cytometry (CytoFLEX S), Laser Confocal Microscope (Nikon A1 Spectral Confocal Microscope), Coy LABORATORY PRODUCTS.
  • CD 11b antibody Novus biologicals, NB 110-89474
  • TNF- ⁇ antibody (CST, 11498);
  • IL-1 ⁇ antibody (CST, 12507);
  • Mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , and cultured in a 37 ° C constant temperature closed incubator (relative humidity 95%). Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments. Hypoxia group Coy cells were placed in hypoxia workstation, the oxygen content is set to 1%, CO 2 content is set at 5%, the rest to fill N 2; with the other normal culture conditions.
  • BV2 cells were seeded in a laser confocal culture plate, and treated with hypoxia for 24 hours, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then washed three times with PBST for 5 minutes each time. After washing, CD 11b antibody diluted with DAKO antibody dilution was added and incubated overnight at 4 °C. The corresponding fluorescently labeled secondary antibody was added to the next day and incubated at 37 ° C for 1 hour. After the incubation, DAPI working solution was added for nuclear staining for 10 minutes. After the end of the treatment, it was washed three times with PBST and imaged using a laser confocal microscope.
  • 2-NBDG uptake assay BV2 cells were seeded in 96-well plate culture plates (black on the sides, transparent on the bottom) and replaced with DPBS buffer containing 200 ⁇ M 2-NBDG after 24 hours, placed in normoxia or hypoxia The cells were cultured for 1 hour, then replaced with fresh DPBS and photographed under a fluorescence microscope and the fluorescence values of the respective treatment groups were detected using a multi-function microplate reader to represent the relative uptake of glucose.
  • Annexin/PI staining After cells were inoculated into a 35 mm culture dish for 24 hours, they were treated with hypoxia for 24 hours. The cells were harvested by digesting with 0.25% trypsin. Then, Annexin and PI dyes were added according to the kit instructions, and 30 minutes later, flow cytometry cells were used to detect whether hypoxia caused cell death.
  • BV2 cells were inoculated to a 60 mm culture dish for 24 hours, and then cultured for 6 hours under normoxia or hypoxia. After washing the cells three times with pre-cooled PBS, 1.5 ml of frozen 80% methanol was added and incubated in a -80 ° C refrigerator for 30 minutes. The cells were collected and centrifuged at 14,000 g for 15 minutes at 4 °C. The upper methanol/water phase was transferred to a new centrifuge tube and incubated for an additional 30 minutes in a 80 °C freezer. The supernatant was collected by centrifugation into a new centrifuge tube, and the sample was blown dry under a nitrogen atmosphere. Samples were stored at -80 °C prior to liquid chromatography-mass spectrometry.
  • hypoxia can induce the morphology of microglia to exhibit a distinct activation state, characterized by an increase in cell body and an increase in pseudopod; and this change is time-dependent and occurs 24 hours after hypoxia.
  • Activated cells account for approximately 52% of total cells (as shown in Figure 1B).
  • Fig. 1C it can be seen that after 24 hours of hypoxia, the activated molecular marker CD 11b of microglia was also significantly enhanced.
  • Fig. 1A hypoxia can induce the morphology of microglia to exhibit a distinct activation state, characterized by an increase in cell body and an increase in pseudopod; and this change is time-dependent and occurs 24 hours after hypoxia.
  • Activated cells account for approximately 52% of total cells (as shown in Figure 1B).
  • Fig. 1C it can be seen that after 24 hours of hypoxia, the activated molecular marker CD 11b of microglia was also significantly enhanced.
  • hypoxia As hypoxia progresses, the aerobic glycolysis pathway of BV2 cells is also significantly enhanced. As shown in Figures 1G and 1H, hypoxia for 1 hour resulted in an approximately 1.6-fold increase in glucose uptake by BV2 cells. At the same time, this process is accompanied by an accumulation of metabolites in the glycolysis pathway, as shown by 1I, compared to the normal control group, fructose 1,6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in hypoxia There was a significant increase in the 6-hour treatment group.
  • Example 2 Hypoxia-induced microglial activation involves up-regulation of hexokinase family members
  • MATERIALS Mouse BV2 microglia cell line, primary cultured mouse microglia, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), RNA extraction reagent TRIzol ( Thermo Fisher Scientific, 15596-018), RNA quantification kit (Thermo Fisher Scientific, Q10211), SuperReal qPCR PreMix (SYBR Green) (Tiangen, FP202-01), real-time PCR instrument (Applied Biosystems), anoxic workstation ( Coy LABORATORY PRODUCTS).
  • Hexokinase 1 antibody (Abcam, 150423);
  • Hexokinase 3 antibody (Santa Cruz, sc-28890);
  • the primer information used for real-time PCR is as follows:
  • Mouse HK1 forward primer GTAGGGGTACGCTTAGGTGG;
  • Mouse HK1 reverse primer ACCCAGGAGTCCATAAAGCC;
  • Mouse HK2 forward primer GAGAAAGCTCAGCATCGTGG;
  • Mouse HK2 reverse primer TCCATTTGTACTCCGTGGCT;
  • Mouse HK3 forward primer GCTCCGTTGAGAGCAGATTT
  • Mouse HK3 reverse primer TTGCTGCAAGCATTCCAGTT
  • Mouse PFKM forward primer GTTTGAGAGCCTCTCCTCCTC;
  • Mouse PFKL forward primer CGCAAGGTATGAATGCTGCT;
  • Mouse PFKL reverse primer CGATGGTCAAGTGTGCGTAG;
  • Mouse PGK1 forward primer CGAGCCTCACTGTCCAAACT;
  • Mouse PGK1 reverse primer GGTCGACACTTTAGCGCCTC;
  • Mouse PKM1 forward primer CGTCCGCAGGTTTGATGAGA;
  • Mouse PKM2 forward primer GGCTCCTATCATTGCCGTGA;
  • Mouse Actb reverse primer TTTGGGGGATGTTTGCTCCA.
  • Mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , and cultured in a 37 ° C constant temperature closed incubator (relative humidity 95%). Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • mice C57BL/6J suckling mice were isolated from 0-2 days after birth, as follows: the cerebral cortex was removed, the meninges and blood vessels were removed; the tissue pieces were cut with tissue scissors, and 0.125% trypsin was used. Digest for 15 minutes at 37 °C. The digestion was stopped in DMEM containing 10% FBS, and the isolated single cells were seeded in a Petri dish. When the mixed culture cells were grown to the fusion, the culture cells were gently shaken to separate the suspended microglia. The purity of the cells was determined using the microglia-specific molecular marker CD 11b.
  • the glycolysis pathway metabolic enzymes were up-regulated at different mRNA levels after 6 hours of hypoxia treatment in BV2 cells compared with the normal control group. Among them, hexokinase 1, hexokinase 2 and hexokinase 3 were significantly elevated. In addition, BV2 cells and primary cultured microglia cells can also be seen transiently or continuously up-regulated at the protein level in Figures 2B and 2C after different periods of hypoxia treatment.
  • Hexokinase 2 interference is effective in inhibiting microglia induced by hypoxia Activation process
  • MATERIALS Mouse BV2 microglia cell line, primary cultured mouse microglia, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), siRNA fragment, siRNA transfer Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, 13778-500), Nikon ECLIPSE Ti Microscope, Nikon A1 Spectral Confocal Microscope, Coy LABORATORY PRODUCTS.
  • Hexokinase 1 antibody (Abcam, 150423);
  • Hexokinase 3 antibody (Santa Cruz, sc-28890);
  • CD 11b antibody Novus biologicals, NB 110-89474
  • Mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , and cultured in a 37 ° C constant temperature closed incubator (relative humidity 95%). Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • RNA interference After cells were inoculated into 35 mm culture for 24 hours, 50 nM siRNA fragments were transfected with RNAiMAX, scrambled RNA was used as a control, and replaced with fresh medium after 12 hours. Hypoxia treatment was then administered for 24 hours to detect the expression level of the corresponding protein.
  • BV2 cells transfected into different interference fragments of HK2, which can effectively inhibit the morphological changes of microglia induced by hypoxia; and up-regulate the label of CD 11b induced by hypoxia. There was also a significant reduction in expression. In the process, the interference of HK2 has no effect on the expression of HK1 and HK3.
  • MATERIALS Mouse BV2 microglia cell line, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), siRNA fragment, siRNA transfection reagent (Lipofectamine RNAiMAX Reagent, Thermo Fisher Scientific) , 13778-500), inverted phase contrast microscope (Nikon ECLIPSE Ti Microscope), anoxic workstation (Coy LABORATORY PRODUCTS). Immunoblotting used antibody information: Hexokinase 1 antibody (Abeam, 150423); Hexokinase 3 antibody (Santa Cruz, sc-28890); ⁇ -Tubulin antibody (Bioworld, AP0064).
  • Mouse BV2 microglial cell lines were grown in high glucose DMEM medium containing 10% FBS, and placed in 5% CO 2, 37 °C closed thermostatic incubator (relative humidity 95%) of the culture Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • RNA interference After cells were inoculated into 35 mm culture for 24 hours, 50 nM siRNA fragments were transfected with RNAiMAX, scrambled RNA was used as a control, and replaced with fresh medium after 12 hours. Hypoxia treatment was then administered for 24 hours to detect the expression level of the corresponding protein.
  • PLM2 Pyruvate kinase M2 subtype
  • MATERIALS Mouse BV2 microglia cell line, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), siRNA fragment, siRNA transfection reagent (Lipofectamine RNAiMAX Reagent, Thermo Fisher Scientific) , 13778-500), inverted phase contrast microscope (Nikon ECLIPSE Ti Microscope), anoxic workstation (Coy LABORATORY PRODUCTS). Immunoblotting used antibody information: PKM2 antibody (Abeam, 150423); CD 11b antibody (Novus biologicals, NB 110-89474); ⁇ -Tubulin antibody (Bioworld, AP0064).
  • Mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , and cultured in a 37 ° C constant temperature closed incubator (relative humidity 95%). Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • RNA interference After cells were inoculated into 35 mm culture for 24 hours, 50 nM siRNA fragments were transfected with RNAiMAX, scrambled RNA was used as a control, and replaced with fresh medium after 12 hours. Hypoxia treatment was then administered for 24 hours to detect the expression level of the corresponding protein.
  • PKM2 protein showed a transient upward expression during hypoxia-induced microglia activation; however, the use of PKM2 interference fragment to interfere with PKM2 expression was not effective in inhibiting hypoxia. Morphological changes associated with activation of microglia (shown in Figures 5B and 5C).
  • the hexokinase 2 inhibitor lonidamine can effectively inhibit the activation process of microglia induced by hypoxia
  • MATERIALS Mouse BV2 microglia cell line, primary cultured mouse microglia, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), lonidamine ( Selleck, S2610), inverted phase contrast microscope
  • Mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , and cultured in a 37 ° C constant temperature closed incubator (relative humidity 95%). Passage, observe growth under an inverted microscope. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • mice C57BL/6J suckling mice were isolated from 0-2 days after birth, as follows: the cerebral cortex was removed, the meninges and blood vessels were removed; the tissue pieces were cut with tissue scissors, and 0.125% trypsin was used. Digest for 15 minutes at 37 °C. The digestion was stopped in DMEM containing 10% FBS, and the isolated single cells were seeded in a Petri dish. When the mixed culture cells were grown to the fusion, the culture cells were gently shaken to separate the suspended microglia. The purity of the cells was determined using the microglia-specific molecular marker CD 11b.
  • BV2 cells and primary cultured microglia were seeded in a laser confocal culture plate, and treated with hypoxia for 24 hours, fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then PBST was used. Wash three times for 5 minutes each time. After the end of the washing, the CD 11b antibody diluted with the DAKO antibody dilution was added and incubated at 4 ° C overnight. The corresponding fluorescently labeled secondary antibody was added to the next day and incubated at 37 ° C for 1 hour. After the incubation, DAPI working solution was added for nuclear staining for 10 minutes. After the end of the treatment, it was washed three times with PBST and imaged using a laser confocal microscope.
  • CD 11b in BV2 cells and primary microglia, the protein expression of CD 11b was significantly up-regulated 24 hours after hypoxia treatment, and a significant activation-like morphology was observed.
  • the up-regulated expression of CD 11b was significantly inhibited in the presence of the glycokinase 2 inhibitor lonidamine, whereas the solvent control group added to DMSO had no effect on the expression of CD 11b.
  • Lonidamine was used at a dose of 50 ⁇ M and DMSO as its solvent.
  • Example 4 Up-regulated expression of hexokinase 2 results in an increase in histone acetylation and transcriptional expression of the pro-inflammatory cytokine Il1b.
  • MATERIALS Mouse BV2 microglia cell line, primary cultured mouse microglia, high glucose DMEM medium (Gibco, 11965-118), fetal bovine serum (Gibco, 10099-141), lonidamine ( Selleck, S2610), 3-bromopyruvate (Sigma, 16490), RNA extraction reagent TRIzol (Thermo Fisher Scientific, 15596-018), RNA quantification kit (Thermo Fisher Scientific, Q10211), SuperReal qPCR PreMix (SYBR Green) ( Tiangen, FP202-01), real-time PCR instrument (Applied Biosystems), chromatin immunoprecipitation kit (Millipore, 17-245), Coy LABORATORY PRODUCTS, inverted phase contrast microscope (Nikon ECLIPSE Ti Microscope).
  • Immunoblotting used antibody information: acetyl-Histone H3 antibody (Millipore, 06-599); acetyl-Histone H4 antibody (Millipore, 06-866); ⁇ -Tubulin antibody (Bioworld, AP0064).
  • the mouse microglia cell line BV2 was grown in high glucose DMEM medium containing 10% FBS, placed in 5% CO 2 , cultured in a 37 ° C constant temperature incubator (relative humidity 95%), under inverted microscope Observe the growth. Passage was performed approximately 2-3 days, and logarithmic growth phase cells were used for formal experiments.
  • mice C57BL/6J suckling mice were isolated from 0-2 days after birth, as follows: the cerebral cortex was removed, the meninges and blood vessels were removed; the tissue pieces were cut with tissue scissors, and 0.125% trypsin was used. Digest for 15 minutes at 37 °C. The digestion was stopped in DMEM containing 10% FBS, and the isolated single cells were seeded in a Petri dish. When the mixed culture cells were grown to the fusion, the culture cells were gently shaken to separate the suspended microglia. The purity of the cells was determined using the microglia-specific molecular marker CD 11b.
  • BV2 cells and primary culture microglia were inoculated into 35 mm culture for 24 hours, and then treated with hypoxia for the corresponding time. The total cellular protein is then extracted to detect the expression of acetylated histone protein.
  • BV2 cells were inoculated to a 60 mm culture dish for 24 hours, and then cultured for 6 hours under normoxia or hypoxia. After washing the cells three times with pre-cooled PBS, 1.5 ml of frozen 80% methanol was added and incubated in a -80 ° C refrigerator for 30 minutes. The cells were collected and centrifuged at 14,000 g for 15 minutes at 4 °C. The upper methanol/water phase was transferred to a new centrifuge tube and incubated for an additional 30 minutes in a 80 °C freezer. The supernatant was collected by centrifugation into a new centrifuge tube, and the sample was blown dry under a nitrogen atmosphere. Samples were stored at -80 °C prior to liquid chromatography-mass spectrometry.
  • BV2 cells were seeded in 100 mm culture dishes for 24 hours, pretreated with DMSO or lonidamine After 1 hour, anoxic treatment was given for 6 hours. After the end of the anoxic treatment, a formaldehyde solution (Sigma, F8775) was added to the cell culture medium to a final concentration of 1%, and cross-linking was carried out for 10 minutes. The cells were collected and the DNA was disrupted to 100-1000 bp using a sonicator. Dilute the sample and take 10% as input.
  • the remaining samples were incubated with acetylated histone H3 or acetylated histone H4 antibody overnight at 4 °C.
  • an equal amount of normal rabbit-derived IgG was used as a negative control.
  • protein G-agarose was added for 2 hours.
  • the sample was then washed with a washing solution and incubated in a 0.2 M NaCl solution at 65 ° C for 4 hours to decrosslink.
  • a subsequent quantitative PCR reaction was carried out to detect the amount of binding of Il1b.
  • the primer sequences of the Il1b promoter region are as follows:
  • Fig. 7A compared with the normal control group, the glycolytic pathway, the tricarboxylic acid cycle pathway and the pentose phosphate pathway metabolites in the hypoxic treatment group were changed to varying degrees, and the hypoxia caused acetyl-coenzyme. The accumulation of A was most pronounced; at the same time, this change was inhibited by the inhibitor of hexokinase 2, chloridamine (using dose: 50 ⁇ M) and 3-bromopyruvate (using dose: 10 ⁇ M) (as shown by 7B).
  • hexokinase 2 chloridamine
  • 3-bromopyruvate using dose: 10 ⁇ M
  • acetylated histone H3 and acetylated histone H4 in BV2 and primary microglia also showed a tendency to transiently up-regulate or continuously up-regulate expression with prolonged hypoxia. .
  • Example 5 Inhibition of Hexokinase 2 effectively protects rats from brain damage caused by midbrain artery infarction by inhibiting microglia-mediated neuroinflammation.
  • Lonidamine can effectively protect rats from brain damage caused by middle cerebral artery infarction
  • MATERIALS Lonidamine (Selleck, S2610), healthy male SPF Spague-Dawley (SD) rat, 2,3,5-TriphenylTetrazolium Chloride (TTC) ) (analytically pure), chloral hydrate (analytical grade) (purchased from Tianjin Komi Chemical Reagent Co., Ltd.), MCAo nylon tether.
  • MCAO Middle Cerebral Artery Occlusion
  • the right external carotid artery was dissected, the distal end was ligated, and a loose knot was made between the right common carotid artery bifurcation and the anterior external carotid artery ligature.
  • the right internal carotid artery was dissected and clamped with a microvascular clamp. Use a microscopic ophthalmic surgical scissors to cut a small opening between the two ligatures, insert the nylon suture down to the common carotid artery, tighten the loose knot, and cut it under the ligation of the distal carotid artery.
  • the right external carotid artery was evacuated from the microvascular clip at the internal carotid artery, and the insertion end of the wire was placed at the bifurcation of the right common carotid artery. Pull the external carotid artery outward and downward so that it is in line with the internal carotid artery. Insert the thread into the direction of the internal carotid artery until the resistance is felt. To prevent bleeding, tighten the thread. The microvascular clip was withdrawn and the incision was sutured. Two hours after the operation, lornidamine or the corresponding solvent control 10 mg/kg was administered, and then the wire brush was pulled out, and the cerebral infarction volume was measured 24 hours after the perfusion.
  • Fig. 8A after TTC staining, the brain tissue of the model group and the brain of the solvent control group showed obvious infarct area after cerebral ischemia for 2 hours and reperfusion for 24 hours, compared with the corresponding brain of the drug treatment group. The degree of infarction in the area was significantly reduced. Statistical analysis of the cerebral infarction volume showed that 10 mg/kg of lonidamine had a significant protective effect on ischemic brain damage caused by middle cerebral artery infarction compared with the vehicle control group (Fig. 8B).
  • MATERIALS Lonidamine (Selleck, S2610), healthy male SPF Spague-Dawley (SD) rat, 2,3,5-TriphenylTetrazolium Chloride (TTC) ) (analytically pure), chloral hydrate (analytical grade) (purchased from Tianjin Komio Chemical Reagent Co., Ltd.), MCAo nylon suture, recombinant adeno-associated virus serotype 9 (rAAV-shHK2) carrying shHK2 fragment, Recombinant adeno-associated virus serotype 9 (rAAV-shNC) carrying a scrambled control fragment, immunohistochemistry kit (Abcam, ab80436), brain stereotaxic instrument, inverted phase contrast microscope (Nikon ECLIPSE Ti Microscope), laser confocal microscope (Nikon A1 Spectral Confocal Microscope).
  • MCAO Middle Cerebral Artery Occlusion
  • the right external carotid artery was dissected, the distal end was ligated, and a loose knot was made between the right common carotid artery bifurcation and the anterior external carotid artery ligature.
  • the right internal carotid artery was dissected and clamped with a microvascular clamp. Use a microscopic ophthalmic surgical scissors to cut a small opening between the two ligatures, insert the nylon suture down to the common carotid artery, tighten the loose knot, and cut it under the ligation of the distal carotid artery.
  • the right external carotid artery was evacuated from the microvascular clip at the internal carotid artery, and the insertion end of the wire was placed at the bifurcation of the right common carotid artery. Pull the external carotid artery outward and downward so that it is in line with the internal carotid artery. Insert the thread into the direction of the internal carotid artery until the resistance is felt. To prevent bleeding, tighten the thread. The microvascular clip was withdrawn and the incision was sutured.
  • rAAV9 virus Injecting rAAV9 virus into the striatum of the brain: The virus was packaged in 293T cells, and the titer of qPCR was 3.2 ⁇ 10 12 -3.5 ⁇ 10 12 VG/ml.
  • the interference sequence for HK2 used was: 5'-GCGCAACATTCTCATCGATTT-3';5'-AAATCGATGAGAATGTTGCGC-3'; the scrambled control shRNA sequence was TTCTCCGAACGTG TCACGT.
  • the above-mentioned viruses were injected into the striatum of the brain using a brain stereotaxic apparatus.
  • the specific coordinates were: 1.0 mm in front of the anterior humerus, 3.0 mm in the right side of the midline, and 4.5 mm in the subdural.
  • the volume of the injected virus was 2 ⁇ L, and the injection speed was 0.2 ⁇ L per minute. After the injection, the microinjection stayed at the injection site for 5 minutes before being pulled out.
  • the body weights of the two groups were weighed and there was no significant difference after statistical analysis (the body weights of the two groups were 273.2 ⁇ 6.9 g and 269.3 ⁇ 5.0 g, respectively).
  • the body weights of the two groups were 273.2 ⁇ 6.9 g and 269.3 ⁇ 5.0 g, respectively.
  • three out of each group were selected and the whole brain tissue was taken out.
  • the distribution of the virus in the brain was detected according to the expression of eGFP.
  • the 9A imaging results showed that the virus had a good tissue distribution in the brain. Staining of the sections after surgery showed that the infarcted side hexokinase 2 was up-regulated significantly compared with the normal control.
  • the striatum region Iba-1 staining showed that the cerebral infarct hemisphere in the rAAV9-shNC group, the microglia protuberances shrank and showed an activated morphology; while in the shHK2 group, the microglia showed a normal morphology. And the expression of Iba-1 also decreased significantly.

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Abstract

提供了一种己糖激酶-2特异性抑制剂在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。

Description

己糖激酶2特异性抑制剂在急性中枢神经系统损伤疾病中的应用 技术领域
本发明属于生物医药领域，涉及己糖激酶2特异性抑制剂等在防治急性中枢神经系统损伤疾病中的应用。
背景技术
脑卒中又称脑中风，是一种急性脑血管疾病，是由于脑部血管突然破裂或因血管堵塞导致脑血流减少而引起脑组织损伤的一种疾病，包括缺血性和出血性脑中风，其中缺血性脑中风占总病例的85％左右。
目前组织型纤溶酶原激活剂(t-PA)是FDA批准用于抗中风的药物。但t-PA只适用于中风发生后的3-6小时内，同时患者接受治疗后也存在脑出血和脑水肿的风险，这些缺陷使得t-PA的应用十分受限，并且受益患者也非常少。因此安全有效的能用于急性缺血性脑中风防治的药物显得备受期待。
急性缺血性脑卒中后免疫系统介导的炎症反应是一个被广泛研究的治疗靶点。但目前以此机制为治疗靶点的药物在临床试验中的结果并不理想。例如：芬戈莫德(Fingolimod)和那他珠单抗(Natalizumab)这些针对外周免疫系统炎症反应的药物尽管可有效抑制淋巴细胞向脑实质的渗透，但临床试验结果显示接收治疗后并不能使得中风患者群体受益。因此，对缺血后小胶质细胞所介导的中枢神经系统炎症反应进行深入探索可望为急性缺血性脑卒中的防治提供新的治疗靶点和策略。
发明内容
本发明的发明人通过筛选获得了一系列糖酵解通路基因，鉴定出己糖激酶2的选择性上调介导了缺氧诱导的小胶质细胞的激活过程。发明人证实，具有己糖激酶2选择性抑制活性的一系列生物活性物质可抑制缺氧诱导的小胶质细胞的激活过程。而且，发明人还发现，己糖激酶1和己糖激酶3干扰不能抑制缺氧诱导的小胶质细胞的激活过程。
因此，本发明一方面提供己糖激酶2特异性抑制剂在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。
本发明另一方面提供包含己糖激酶2特异性抑制剂的组合物在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。
本发明再一方面提供一种预防和治疗急性中枢神经系统损伤疾病的方法，所述方法包括向有需要的对象施用预防有效量或治疗有效量的己糖激酶2特异性抑制剂或包含己糖激酶2特异性抑制剂的组合物。
本发明发现了己糖激酶2特异性抑制剂在防治急性中枢神经系统损伤疾病中的神经保护作用。细胞学实验和体内动物实验表明选择性己糖激酶2上调表达调控了缺氧诱导的小胶质细胞的激活过程以及缺血后小胶质细胞介导的神经炎症反应，而己糖激酶2选择性抑制剂和基因敲低都可显著抑制小胶质细胞介导的炎症反应从而发挥神经保护作用。
附图说明
图1：糖酵解通路的增强对于缺氧所引起的小胶质细胞的激活是必须的。(A)暴露于缺氧环境所示时间后BV 2小胶质细胞的代表性图像。比例尺100μm(n＝4)。(B)图(A)中具有形态学变化的小胶质细胞百分数的定量(n＝4)。(C)缺氧引起的小胶质细胞的激活通过分子标记物CD11b的上调而确认。比例尺25μm(n＝3)。(D)BV 2细胞缺氧显著诱导促炎细胞因子的产生且呈时间依赖性(n＝3)。(E–F)缺氧对BV 2细胞的存活率没有影响。流式细胞分析显示，在暴露于1％氧气24h后AnnexinV +或PI +细胞的数量没有显著增加(n＝3)。(G)在与2-[N-(7-硝基苄基-2-氧杂-1,3-二唑-4-基)氨基]-2-脱氧-D-葡萄糖(2-NBDG)共培养并暴露于常氧或缺氧1h的细胞中2-NBDG摄取试验的代表性图片。比例尺100μm。(H)使用多点检测酶标仪定量(G)中的平均荧光强度(n＝4)。(I)糖酵解途径中关键代谢物的图示；标记出暴露于缺氧6h时后显著增强的代谢物(n＝4)。(J-L)糖酵解途径的抑制剂2-DG显著阻断缺氧期间的小胶质细胞激活。(J)当暴露于1％氧气时，添加或不添加2-DG的BV 2细胞的图像对比。(K)在(J)中用2-DG处理后激活细胞的百分比下降。比例尺50μm(n＝4)。(L)在缺氧条件下促炎细胞因子的生成被2-DG显著破坏。*<0.05；**<0.01；***<0.001。
图2：缺氧诱导的小胶质细胞激活过程涉及到己糖激酶家族成员的上调表达。(A)qRT-PCR分析酶的mRNA水平。图片显示在缺氧6h后这些基因的mRNA水平总体上增加(n＝3)。(B-C)在缺氧所示时间段后，BV 2细胞(B)和初级小胶质细胞(pMG)(C)中的HK1、HK2和HK3蛋白水平(n＝4)。
图3：己糖激酶2干扰能有效抑制由缺氧诱导的小胶质细胞的激活过程。(A)特异性的HK2敲低(knockdown)足以阻断BV 2小胶质细胞的激活表型(n＝3)。使用或不使用HK2siRNA转染BV 2细胞24h并在缺氧条件下刺激另外24h后，Western杂交分析所示蛋白的水平。(B)BV2细胞转染入HK2不同的干扰片段后，能有效抑制由缺氧诱导的小胶质细胞形态学改变。(C)HK2敲低细胞在缺氧条件下激活形态的小胶质细胞的百分比降低。(D)HK2敲低显著抑制CD11b的表达。
图4：己糖激酶1和己糖激酶3干扰不能抑制缺氧诱导的小胶质细胞的激活过程。使用HK1和HK3干扰片段分别干扰己糖激酶1和己糖激酶3后(A和C)，并不能有效抑制由缺氧引起的小胶质细胞的激活相关的形态学改变(B和D)(n＝3)。
图5：丙酮酸激酶M2亚型(PKM2)干扰不能抑制缺氧诱导的小胶质细胞的激活过程。(A)缺氧刺激的BV 2细胞中PKM2蛋白表达的免疫印迹分析。(B)PKM2敲低不影响缺氧诱导的CD11b的上调。(C)代表性图片显示PKM2敲低无法抑制缺氧诱导的形态学改变。比例尺50μm。
图6：己糖激酶2抑制剂氯尼达明能有效抑制由缺氧诱导的小胶质细胞的激活过程。(A)缺氧期间pMG和BV 2培养物的激活状态在存在氯尼达明(50μM)的情况下被显著抑制。比例尺50μm。(B)图片显示在(A)中用氯尼达明处理后激活的小胶质细胞的百分比降低。(C)免疫荧光试验显示缺氧刺激下的BV 2和pMG细胞在存在氯尼达明时CD11b表达降低。比例尺50μm。*<0.05；**<0.01；***<0.001。
图7：己糖激酶2的上调表达导致组蛋白乙酰化的增加和促炎细胞因子Il1b的转录表达。(A)BV 2细胞暴露于缺氧条件下6h后糖酵解和TCA循环的代谢物图谱。数据表示缺氧对常氧的变化倍数。下调代谢物以绿色方块表示，上调代谢物以红色方块表示(n＝4)。(B)(D)HK2抑制逆转了BV 2细胞的细胞内乙酰辅酶A的积聚并抑制乙酰化组蛋白的上调。(C)BV 2细胞和初级小胶质细胞(pMG)在缺氧暴露后组蛋白乙酰化的表达水平(n＝3)。(E)mRNA水平的Il1b的缺氧诱导的上调可通过HK2抑制显著降低(n＝3)。(F)氯尼达明预处理降低了AcH3和AcH4与Il1b启动子的结合。每个处理组中的具有AcH3或AcH4的Il1b启动子的丰度是相对于具有相同引物的相应输入样品而言的(n＝3)。*<0.05；**<0.01；***<0.001。
图8：氯尼达明有效保护大鼠免于中脑动脉梗塞所造成的脑损伤。(A)每个处理组中2,3,5-氯化三苯基四氮唑(TTC)染色的脑切片的代表性图片。(B)每组中脑梗死大小的定量，显示氯尼达明给药组显著降低MCAo造成的梗死大小(n＝6/组)。*<0.05；**<0.01；***<0.001。
图9：体内基因敲低己糖激酶2有效保护大鼠免于中脑动脉梗塞所造成的脑损伤。(A)在注射AAV载体21天后，利用全脑成像基于eGFP表达来监测病毒分布(n＝3)。(B)大脑纹状区切片的HK2和CD 11b染色显示CD 11b在小胶质细胞中与HK2表达呈正相关(n＝6/组)。比例尺20μm。(C)TTC染色显示MCAo手术后用AAV shHK2处理的组中梗死面积减小(n＝6/组)。(D)图(C)中梗死大小的定量(n＝6/组)。(E)纹状体Iba-1免疫反应性试验显示AAV-shHK2处理显著抑制梗死的脑半球的小胶质细胞的激活。比例尺50μm。(F)缺血脑半球的皮质和纹状区的代表性图像，显示在大鼠MCAo模型中AAV9-shHK2处理后IL-1b的产生减少。
具体实施方式
以下实施方式是对本发明作进一步说明，但本发明的实施方式不局限于以下的实施例介绍，凡依照本发明的原理或理念所作的等同的变化或变通都应视为本发明保护的范畴。
缩略词
2-NBDG，2-[N-(7-硝基苯-2-氧杂-1,3-二唑-4-基)氨基]-2-脱氧-D-葡萄糖；
ChIP，染色质免疫沉淀；
DAB，3,3'-二氨基联苯胺四盐酸盐；
DAMP，损伤相关的分子模式；
DHAP，磷酸二羟丙酮；
FBP，果糖-1,6-二磷酸；
G-3-P，甘油醛3-磷酸；
HK2，己糖激酶2；
IL-1β，白介素-1β；
LPS，脂多糖；
MCAo，大脑中动脉闭塞；
NMDA，N-甲基-D-天冬氨酸；
PKM2，丙酮酸激酶M2；
qRT-PCR，定量逆转录聚合酶链式反应；
rAAV，重组腺相关病毒；
ROS，活性氧；
siRNA，短干扰RNA；
TTC，2,3,5-三苯基氯化四氮唑。
在一个方面，本发明提供一种己糖激酶2特异性抑制剂在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。所述己糖激酶2特异性抑制剂是指能够特异性或选择性抑制己糖激酶2(也称为己糖激酶II或HK2)的生物学活性的物质。
在一些实施方式中，所述己糖激酶2特异性抑制剂包括己糖激酶2的抗体或其片段。所述抗体是指能够特异性结合至己糖激酶2并抑制或淬灭己糖激酶2的活性的蛋白质。抗体的片段可包括例如Fab、Fab'、(Fab') 2和Fv。抗体或其片段的生产及纯化在本领域是已知的。
在一些实施方式中，本发明所述的己糖激酶2抗体还可以编码该抗体的氨基酸序列、核苷酸序列、包含该核苷酸序列或氨基酸序列的表达载体的形式存在。在一些实施方式中，本发明所述的己糖激酶2抗体还可以融合蛋白的形式存在于表达载体或宿主细胞中。
在一些实施方式中，所述己糖激酶2特异性抑制剂包括能够特异性抑制己糖激酶2的mRNA翻译的物质，或能够特异性降解己糖激酶2的mRNA的物质，例如siRNA，shRNA、miRNA或其修饰物，从而通过RNAi机制干扰己糖激酶2的合成。siRNA，shRNA或miRNA可通过体外合成技术获得，这在本领域是熟知的。在一些实施方式中，本发明所述siRNA，shRNA或miRNA存在于特定载体中，例如细胞中。
在一些实施方式中，所述急性中枢神经系统损伤疾病是指由急性缺血或缺氧引起的中枢神经系统损伤的疾病或病症，包括但不限于，急性脊柱损伤、脑外伤、视网膜损伤、缺氧性脑损伤、急性缺血性脑损伤、缺血性脑卒中、缺氧性脑卒中、新生儿缺氧缺血性脑病、中毒性脑病、急性脑梗塞、腔隙性脑梗死、短暂性脑缺血发作、重型颅脑损伤、脑脊髓手术和脑脊髓放疗等导致的脑、脊髓神经损伤。
在另一个方面，本发明提供包含己糖激酶2特异性抑制剂的组合物在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。
在一些实施方式中，所述组合物中所包含的己糖激酶2特异性抑制剂如上所述，并且该组合物还可包含其他己糖激酶2抑制剂。所述其他己糖激酶2抑制剂包括但不限于，2-脱氧葡萄糖；氯尼达明(Lonidamine)；3-溴丙酮酸(bromopyruvic acid)；6-磷酸葡萄糖(glucose 6-phosphate)；伊马替尼(Imatinib)；5-硫代葡萄糖(5-thio-glucose)；茉莉酮酸甲酯(methyl jasmonate)。
本发明再一方面提供一种预防和治疗急性中枢神经系统损伤疾病的方法，所述方法包括向有需要的对象施用预防有效量或治疗有效量的己糖激酶2特异性抑制剂或包含己糖激酶2特异性抑制剂的组合物。
在一些实施方式中，所述对象是指哺乳动物对象，例如人类。所述施用是指通过皮下、经皮、肌内、静脉内、动脉内、舌下、口腔、胃肠道等给药方式将药物用于例如人体。在一些实施方式中，可通过基因疗法将己糖激酶2特异性抑制剂(例如核酸形式)施用至被治疗或预防的对象。
本发明再一方面提供一种预防和治疗急性中枢神经系统损伤疾病的方法，所述方法包括有此需要的对象体内选择性或特异性降低或灭活己糖激酶2的活性。所述降低或灭活己糖激酶的活性可通过例如基因工程手段降低或消除己糖激酶2蛋白质的表达来实现。一个示例性 的手段为通过定点突变改变己糖激酶2编码核苷酸的序列。另一个示例性的手段为通过RNAi技术干扰己糖激酶2的mRNA的翻译过程。任何本领域技术人员已知的在细胞内降低特定蛋白质表达的方法预期都可以用于本发明的方法中。
在没有特别指明的情况下，本发明采用的材料及实验方法为常规材料及方法。
实施例1 缺氧所引起的糖酵解通路的增强对于小胶质细胞的激活是必须的
材料：小鼠BV2小胶质细胞株，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，2-脱氧葡萄糖(Sigma-Aldrich,D8375)，2-(N-7-硝基-2,1,3-苯并二唑-4-氨基)-2-脱氧葡萄糖(2-NBDG，Thermo Fisher Scientific,N13195)，Annexin/PI染色试剂盒(Biotool,B32115),流式细胞分析仪(CytoFLEX S)，激光共聚焦显微镜(Nikon A1Spectral Confocal Microscope)，缺氧工作站(Coy LABORATORY PRODUCTS)。
免疫印迹以及免疫荧光使用抗体信息：
CD 11b抗体(Novus biologicals,NB 110-89474)；
TNF-α抗体(CST,11498)；
IL-1β抗体(CST,12507)；
IL-6抗体(Bioss,bs-6309R)；
α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。缺氧处理组细胞置于Coy缺氧工作站中，氧气含量设置为1％，CO 2含量设置为5％，其余以N 2填充；其他同正常培养条件。
b)细胞蛋白提取与促炎细胞因子蛋白表达检测：细胞接种至35mm培养基中静置24小时后，给以缺氧处理相应时间。之后提取细胞总蛋白检测促炎细胞因子TNF-α，IL-1β和IL-6的蛋白表达。
c)免疫荧光检测CD 11b表达：BV2细胞接种于激光共聚焦专用培养板中，给予缺氧处理24小时后使用4％多聚甲醛室温固定20分钟，之后使用PBST洗涤三次，每次5分钟。洗涤结束后，加入由DAKO抗体稀释液稀释过的CD 11b抗体在4℃孵育过夜。次日加入对应的荧光标记二抗37℃孵育1小时。孵育结束后，加入DAPI工作液进行细胞核染色10分钟。处理结束后，以PBST洗涤三次并利用激光共聚焦显微镜进行成像。
d)2-NBDG摄取实验：BV2细胞接种于96孔板培养板(侧边黑色，底部透明)中，24小时后更换为含有200μM 2-NBDG的DPBS缓冲液，分别置于常氧或者缺氧环境中培养1小时，之后更换为新鲜的DPBS在荧光显微镜下拍照并利用多功能酶标仪检测各个处理组的荧光值以代表葡萄糖的相对摄取量。
e)Annexin/PI染色：细胞接种至35mm培养皿中静置24小时后，给以缺氧处理24小时。用0.25％胰酶消化收集细胞。之后按照试剂盒说明书加入Annexin和PI染料，30分钟后利用流式细胞仪细胞检测缺氧是否引起细胞的死亡。
f)代谢物质的检测：BV2细胞接种至60mm培养皿静置24小时后，给以常氧或者缺氧培养6小时。以预冷PBS洗涤细胞三次后，加入1.5ml冰冻的80％的甲醇，-80℃冰箱中孵育30分钟。收集细胞，4℃14,000g离心15分钟。取上层甲醇/水相转移至新的离心管中-80℃冰箱中再次孵育30分钟。离心收集上清至新的离心管中，在氮气氛围中吹干样品。样品经液相-质谱分析之前置于-80℃保存。
结果：
如图1A所示，缺氧可以诱导小胶质细胞的形态呈现出明显的激活状态，特点为胞体增大，伪足增多；且这种变化具有时间依赖性，在缺氧24小时后，出现激活形态的细胞大约占总细胞的52％左右(如图1B所示)。同时，在图1C免疫荧光的结果中可以看到缺氧24小时后，小胶质细胞被激活的分子标记物CD 11b也有显著增强。另外，如图1D所示，促炎细胞因子TNF-α，IL-1β和IL-6在正常对照组几乎无表达，随着缺氧时间的延长这些蛋白的表达量也有明显增加。而在整个缺氧过程中，并没有发现1％的缺氧环境会造成细胞的死亡(如图1E和1F所示)。以上结果表明，缺氧可使得小胶质细胞呈现一种炎性的激活表型。
随着缺氧的进行，BV2细胞的有氧糖酵解通路也明显增强。如图1G和1H所示，缺氧1小时即可以引起BV2细胞葡萄糖摄取量增加约1.6倍。同时，这个过程也伴随着糖酵解通路代谢物的累积增加，如1I所示，相比于正常对照组，1,6-二磷酸果糖，磷酸二羟丙酮和3-磷酸甘油醛在缺氧处理6小时组都有显著的增加。而在使用2-脱氧葡萄糖对糖酵解通路进行抑制后，由缺氧引起的小胶质细胞的激活表型被显著抑制(如图1J-1L所示)。综上所述，糖酵解通路的增强对于缺氧诱导的小胶质细胞的激活是必须的。
实施例2 缺氧诱导的小胶质细胞激活过程涉及到己糖激酶家族成员的上调表达
材料：小鼠BV2小胶质细胞株，原代培养小鼠小胶质细胞，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，RNA提取试剂TRIzol(Thermo Fisher Scientific,15596-018)，RNA定量试剂盒(Thermo Fisher Scientific,Q10211)，SuperReal qPCR PreMix (SYBR Green)(Tiangen,FP202-01)，实时荧光定量PCR仪(Applied Biosystems)，缺氧工作站(Coy LABORATORY PRODUCTS)。
免疫印迹使用抗体信息：
Hexokinase 1抗体(Abcam,150423)；
Hexokinase 2抗体(CST,2867s)；
Hexokinase 3抗体(Santa Cruz,sc-28890)；
α-Tubulin抗体(Bioworld,AP0064)。
实时荧光定量PCR所使用的引物信息如下：
小鼠HK1正向引物:GTAGGGGTACGCTTAGGTGG；
小鼠HK1反向引物:ACCCAGGAGTCCATAAAGCC；
小鼠HK2正向引物:GAGAAAGCTCAGCATCGTGG；
小鼠HK2反向引物:TCCATTTGTACTCCGTGGCT；
小鼠HK3正向引物:GCTCCGTTGAGAGCAGATTT；
小鼠HK3反向引物:TTGCTGCAAGCATTCCAGTT；
小鼠PFKM正向引物:GTTTGGAAGCCTCTCCTCCTC；
小鼠PFKM反向引物:GACGGCAGCATTCATACCTT；
小鼠PFKL正向引物:CGCAAGGTATGAATGCTGCT；
小鼠PFKL反向引物:CGATGGTCAAGTGTGCGTAG；
小鼠PGK1正向引物:CGAGCCTCACTGTCCAAACT；
小鼠PGK1反向引物:GTCTGCAACTTTAGCGCCTC；
小鼠PKM1正向引物:CGTCCGCAGGTTTGATGAGA；
小鼠PKM1反向引物:TTCAAACAGCAGACGGTGGA；
小鼠PKM2正向引物:GGCTCCTATCATTGCCGTGA；
小鼠PKM2反向引物:AAGGTACAGGCACTACACGC；
小鼠Actb正向引物:TGAGCTGCGTTTTACACCCT；
小鼠Actb反向引物:TTTGGGGGATGTTTGCTCCA。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
原代培养小胶质细胞：细胞分离至出生后0-2天的C57BL/6J乳鼠，具体如下：取出大脑皮层，去除脑膜和血管；用组织剪剪碎组织块，用0.125％的胰酶于37℃消化15分钟。以含有10％FBS的DMEM终止消化，分离的单细胞接种于培养皿中。待混合培养细胞长至融合时，轻摇培养皿分离出悬浮的小胶质细胞。细胞的纯度使用小胶质细胞特异性的分子标记物CD 11b确定。
b)细胞蛋白提取与己糖激酶家族蛋白表达检测：细胞接种至35mm培养中静置24小时后，给以缺氧处理相应时间。之后提取细胞总蛋白检测己糖激酶家族的蛋白表达。
结果：
如图2A所示，相比于正常对照组，BV2细胞缺氧处理6小时后糖酵解通路代谢酶在mRNA水平都有不同程度的上调表达。其中，己糖激酶1，己糖激酶2和己糖激酶3都有显著升高。另外，BV2细胞和原代培养小胶质细胞在缺氧处理不同时间后，图2B和2C中也可以看到己糖激酶家族成员在蛋白质水平都有瞬时性或者持续性上调表达。
实施例3 己糖激酶2，而不是其他己糖激酶家族成员介导缺氧诱导的小胶质细胞的激活过程(1)己糖激酶2干扰能有效抑制由缺氧诱导的小胶质细胞的激活过程
材料：小鼠BV2小胶质细胞株，原代培养小鼠小胶质细胞，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，siRNA片段，siRNA转染试剂(Lipofectamine RNAiMAX Reagent，Thermo Fisher Scientific,13778-500)，倒置相差显微镜(Nikon ECLIPSE Ti Microscope)，激光共聚焦显微镜(Nikon A1Spectral Confocal Microscope)，缺氧工作站(Coy LABORATORY PRODUCTS)。
免疫印迹使用抗体信息：
Hexokinase 1抗体(Abcam,150423)；
Hexokinase 2抗体(CST,2867s)；
Hexokinase 3抗体(Santa Cruz,sc-28890)；
CD 11b抗体(Novus biologicals,NB 110-89474)；
α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
b)RNA干扰：细胞接种至35mm培养中静置24小时后，采用RNAiMAX转染50nM siRNA片段，乱序RNA作为对照，12小时后更换为新鲜的培养基。之后给予缺氧处理24小时以检测相应蛋白的表达量。
结果：
如图3A-3D所示，BV2细胞转染入HK2不同的干扰片段后，能有效抑制由缺氧诱导的小胶质细胞形态学改变；同时缺氧引起的细胞激活的标记物CD 11b的上调表达也有明显减少。而在过程中，HK2的干扰对HK1和HK3的表达并没有影响。
(2)己糖激酶1和己糖激酶3干扰不能抑制缺氧诱导的小胶质细胞的激活过程
材料：小鼠BV2小胶质细胞株，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，siRNA片段，siRNA转染试剂(Lipofectamine RNAiMAX Reagent，Thermo Fisher Scientific,13778-500)，倒置相差显微镜(Nikon ECLIPSE Ti Microscope)，缺氧工作站(Coy LABORATORY PRODUCTS)。免疫印迹使用抗体信息：Hexokinase 1抗体(Abcam,150423)；Hexokinase 3抗体(Santa Cruz,sc-28890)；α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
b)RNA干扰：细胞接种至35mm培养中静置24小时后，采用RNAiMAX转染50nM siRNA片段，乱序RNA作为对照，12小时后更换为新鲜的培养基。之后给予缺氧处理24小时以检测相应蛋白的表达量。
结果：
如图4A和4C所示，使用HK1和HK3干扰片段分别干扰己糖激酶1和己糖激酶3后，并不能有效抑制由缺氧引起的小胶质细胞的激活相关的形态学改变(图4B和4D所示)。
(3)丙酮酸激酶M2亚型(PKM2)干扰不能抑制缺氧诱导的小胶质细胞的激活过程
材料：小鼠BV2小胶质细胞株，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，siRNA片段，siRNA转染试剂(Lipofectamine RNAiMAX Reagent，Thermo Fisher Scientific,13778-500)，倒置相差显微镜(Nikon ECLIPSE Ti Microscope)，缺氧工作站(Coy LABORATORY PRODUCTS)。免疫印迹使用抗体信息：PKM2抗体(Abcam,150423)；CD 11b抗体(Novus biologicals,NB 110-89474)；α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
b)RNA干扰：细胞接种至35mm培养中静置24小时后，采用RNAiMAX转染50nM siRNA片段，乱序RNA作为对照，12小时后更换为新鲜的培养基。之后给予缺氧处理24小时以检测相应蛋白的表达量。
结果：
如图5A所示，在缺氧诱导的小胶质细胞的激活过程中，PKM2蛋白呈现出瞬时上升表达的趋势；但使用PKM2干扰片段干扰PKM2的表达后，并不能有效抑制由缺氧引起的小胶质细胞的激活相关的形态学改变(图5B和5C所示)。
3)己糖激酶2抑制剂氯尼达明能有效抑制由缺氧诱导的小胶质细胞的激活过程
材料：小鼠BV2小胶质细胞株，原代培养小鼠小胶质细胞，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，氯尼达明(Selleck，S2610)，倒置相差显微镜
(Nikon ECLIPSE Ti Microscope)，激光共聚焦显微镜(Nikon A1Spectral Confocal Microscope)，缺氧工作站(Coy LABORATORY PRODUCTS)。免疫荧光使用抗体信息：CD 11b抗体(Novus biologicals,NB 110-89474)；α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
原代培养小胶质细胞：细胞分离至出生后0-2天的C57BL/6J乳鼠，具体如下：取出大脑皮层，去除脑膜和血管；用组织剪剪碎组织块，用0.125％的胰酶于37℃消化15分钟。以含有10％FBS的DMEM终止消化，分离的单细胞接种于培养皿中。待混合培养细胞长至融合时，轻摇培养皿分离出悬浮的小胶质细胞。细胞的纯度使用小胶质细胞特异性的分子标记物CD 11b确定。
b)免疫荧光检测CD 11b表达：BV2细胞和原代培养小胶质细胞接种于激光共聚焦专用培养板中，给予缺氧处理24小时后使用4％多聚甲醛室温固定20分钟，之后使用PBST洗涤三次，每次5分钟。洗涤结束后，加入由DAKO抗体稀释液稀释过的CD 11b抗体在4℃孵 育过夜。次日加入对应的荧光标记二抗37℃孵育1小时。孵育结束后，加入DAPI工作液进行细胞核染色10分钟。处理结束后，以PBST洗涤三次并利用激光共聚焦显微镜进行成像。
结果：
如图6A和6B所示，BV2细胞和原代小胶质细胞中，给予缺氧处理24小时后，CD 11b的蛋白表达明显上调，同时也可以观察到呈现明显的激活样形态；但在己糖激酶2抑制剂氯尼达明存在时，CD 11b的上调表达被明显抑制，而加入DMSO的溶剂对照组对CD 11b的表达并没有影响。氯尼达明使用剂量为：50μM，DMSO为其溶剂。
实施例4 己糖激酶2的上调表达导致组蛋白乙酰化的增加和促炎细胞因子Il1b的转录表达。
材料：小鼠BV2小胶质细胞株，原代培养小鼠小胶质细胞，高糖DMEM培养基(Gibco,11965-118)，胎牛血清(Gibco,10099-141)，氯尼达明(Selleck，S2610)，3-溴丙酮酸(Sigma，16490)，RNA提取试剂TRIzol(Thermo Fisher Scientific,15596-018)，RNA定量试剂盒(Thermo Fisher Scientific,Q10211)，SuperReal qPCR PreMix(SYBR Green)(Tiangen,FP202-01)，实时荧光定量PCR仪(Applied Biosystems)，染色质免疫沉淀试剂盒(Millipore，17-245)，缺氧工作站(Coy LABORATORY PRODUCTS)，倒置相差显微镜(Nikon ECLIPSE Ti Microscope)。免疫印迹使用抗体信息：acetyl-Histone H3抗体(Millipore,06-599)；acetyl-Histone H4抗体(Millipore,06-866)；α-Tubulin抗体(Bioworld,AP0064)。
方法：
a)细胞的培养：
小鼠小胶质细胞株BV2生长在含有10％FBS的高糖DMEM培养基中，置于5％CO 2，37℃恒温密闭式孵箱(相对湿度95％)内培养传代，在倒置显微镜下观察生长情况。大约2-3天传代一次，取对数生长期细胞用于正式实验。
原代培养小胶质细胞：细胞分离至出生后0-2天的C57BL/6J乳鼠，具体如下：取出大脑皮层，去除脑膜和血管；用组织剪剪碎组织块，用0.125％的胰酶于37℃消化15分钟。以含有10％FBS的DMEM终止消化，分离的单细胞接种于培养皿中。待混合培养细胞长至融合时，轻摇培养皿分离出悬浮的小胶质细胞。细胞的纯度使用小胶质细胞特异性的分子标记物CD 11b确定。
b)细胞蛋白提取与乙酰化组蛋白表达检测：BV2细胞和原代培养小胶质细胞接种至35mm培养中静置24小时后，给以缺氧处理相应时间。之后提取细胞总蛋白检测乙酰化组蛋白蛋白的表达。
c)代谢物质的检测：BV2细胞接种至60mm培养皿静置24小时后，给以常氧或者缺氧培养6小时。以预冷PBS洗涤细胞三次后，加入1.5ml冰冻的80％的甲醇，-80℃冰箱中孵育30分钟。收集细胞，4℃14,000g离心15分钟。取上层甲醇/水相转移至新的离心管中-80℃冰箱中再次孵育30分钟。离心收集上清至新的离心管中，在氮气氛围中吹干样品。样品经液相-质谱分析之前置于-80℃保存。
d)染色质免疫沉淀实验检测乙酰化组蛋白H3和乙酰化组蛋白H4与Il1b基因启动子区域的结合：BV2细胞接种于100mm培养皿中静置24小时，以DMSO或者氯尼达明预处理1小时后给予缺氧处理6小时。缺氧处理结束后，在细胞培养基中加入甲醛溶液(Sigma，F8775)使之终浓度为1％，交联10分钟。收集细胞，使用超声破碎仪使之DNA断裂成为100-1000bp。稀释样品，取出10％作为input。剩余样品加入乙酰化组蛋白H3或者乙酰化组蛋白H4抗体4℃孵育过夜。同时，等量的正常兔来源的IgG作为阴性对照。次日，加入蛋白G-琼脂糖反应2小时。之后以洗涤液洗涤样品，在0.2M NaCl溶液中65℃孵育4小时以解交联。酚-氯仿法提取DNA后进行后续定量PCR反应以检测Il1b的结合量。Il1b启动子区域的引物序列如下：
正向：5’-AGGTCAAAGGTTTGGAAGCAG-3’；
反向：5’-ATGGAAGTCTGTCTGCTCAGTATTG-3’。
结果：
如图7A所示，相比于正常对照组，缺氧6小时处理组后糖酵解通路，三羧酸循环通路和戊糖磷酸通路代谢物都有不同程度的改变，其中缺氧使得乙酰辅酶A的累积最为明显；同时这种改变可以被己糖激酶2的抑制剂氯尼达明(使用剂量：50μM)和3-溴丙酮酸(使用剂量：10μM)所抑制(如7B所示)。另外，如图7C所示，随着缺氧时间的延长，BV2和原代小胶质细胞中的乙酰化组蛋白H3和乙酰化组蛋白H4也呈现出与瞬时上调表达或者持续上调表达的趋势。
在BV2细胞中，缺氧6小时所导致的乙酰化组蛋白的上调表达也是可以被己糖激酶2的抑制剂所逆转的，图7D所示。进一步对促炎细胞因子的mRNA表达水平进行检测，如7E所示，实时荧光定量PCR结果显示缺氧引起Tnfa，Il1b和Il6的mRNA上调表达，但只有Il1b可明显被氯尼达明(使用剂量：50μM)和3-溴丙酮酸(使用剂量：10μM)所抑制。染色质免疫沉淀结果进一步显示：缺氧后可以使得乙酰化组蛋白和Il1b的启动子结合增加，而氯尼达明预处理显著抑制两者的结合(7F所示)。
实施例5 己糖激酶2的抑制通过抑制小胶质细胞介导的神经炎症有效保护大鼠免于中脑动脉梗塞所造成的脑损伤。
(1)氯尼达明有效保护大鼠免于中脑动脉梗塞所造成的脑损伤
材料：氯尼达明(Selleck，S2610)，健康雄性SPF级Spague-Dawley(SD)大鼠，2,3,5-氯化三苯基四氮唑(2,3,5-TriphenylTetrazolium Chloride，TTC)(分析纯)，水合氯醛(分析纯)(购自天津市科密欧化学试剂有限公司)，MCAo尼龙栓线。
方法：
a)采用右侧颈内动脉尼龙线线栓法制作大脑中动脉阻塞(Middle Cerebral Artery Occlusion,MCAO)模型。SD大鼠术前禁食12h，自由饮水。用10％的水合氯醛腹腔注射麻醉，仰卧位固定于37℃恒温手术台上，保持呼吸通畅。颈正中切口，钝性分离下颌下腺，显微镜下游离右侧颈总动脉，使用微血管夹夹闭。游离右侧颈外动脉，结扎其远端，在右颈总动脉分叉至前一颈外动脉结扎线之间打一松结，游离右颈内动脉并用微血管夹夹闭。用显微眼科手术剪刀在两结扎线之间剪一小口，将尼龙栓线向下插入至颈总动脉处，将松结扎紧，在颈外动脉远端结扎处下方、栓线插入的上方剪断右颈外动脉，撤离颈内动脉处的微血管夹，并使栓线插入端在右颈总动脉分叉处。将颈外动脉拉向外下方，使其与颈内动脉处于同一直线。将栓线向颈内动脉方向插入直至感受到阻力，为防止出血将栓线扎紧。撤离微血管夹，缝合切口。手术2小时后，给予氯尼达明或者相对应溶剂对照10mg/kg，之后拔出线刷，再灌注24小时后进行脑梗死体积测量。
b)根据相关文献介绍方法进行脑梗死体积测量，具体为断头处死大鼠，迅速取出鼠脑，置于冰盐水中10min，取冠状面均匀切成2mm厚脑片，迅速放入1％的TTC溶液中于37℃染色30min，然后用4％多聚甲醛缓冲液固定。24h后用数码相机拍照，输入计算机，用图像处理软件(ADOBE PHOTOSHOP CS6)计算每片脑片的梗死面积(粉红色区为正常脑组织，白色区为梗死区)后积分转换为梗死容积。梗死容积百分比＝每片脑片的梗死区域体积之和/每片脑片的全脑组织体积之和×100％。
结果：
如图8A所示，经TTC染色后观察，脑缺血2小时再灌注24小时后模型组大鼠脑组织和溶剂对照组脑组织都出现明显的梗死区域，与之相比药物处理组相应脑区的梗死程度明显减轻。计算脑梗死体积经过统计学分析显示，与溶剂对照组相比，10mg/kg的氯尼达明对大鼠中脑动脉梗塞造成的缺血脑损伤具有显著保护作用(如图8B)。
(2)体内基因敲低己糖激酶2有效保护大鼠免于中脑动脉梗塞所造成的脑损伤
材料：氯尼达明(Selleck，S2610)，健康雄性SPF级Spague-Dawley(SD)大鼠，2,3,5-氯化三苯基四氮唑(2,3,5-TriphenylTetrazolium Chloride，TTC)(分析纯)，水合氯醛(分析纯)(购自天津市科密欧化学试剂有限公司)，MCAo尼龙栓线，携带shHK2片段的重组腺相关病毒血清型9型(rAAV-shHK2)，携带乱序对照片段的重组腺相关病毒血清型9型(rAAV-shNC)，免疫组织化学试剂盒(Abcam,ab80436)，脑立体定位仪，倒置相差显微镜(Nikon ECLIPSE Ti Microscope)，激光共聚焦显微镜(Nikon A1Spectral Confocal Microscope)。
方法：
a)采用右侧颈内动脉尼龙线线栓法制作大脑中动脉阻塞(Middle Cerebral Artery Occlusion,MCAO)模型。SD大鼠术前禁食12h，自由饮水。用10％的水合氯醛腹腔注射麻醉，仰卧位固定于37℃恒温手术台上，保持呼吸通畅。颈正中切口，钝性分离下颌下腺，显微镜下游离右侧颈总动脉，使用微血管夹夹闭。游离右侧颈外动脉，结扎其远端，在右颈总动脉分叉至前一颈外动脉结扎线之间打一松结，游离右颈内动脉并用微血管夹夹闭。用显微眼科手术剪刀在两结扎线之间剪一小口，将尼龙栓线向下插入至颈总动脉处，将松结扎紧，在颈外动脉远端结扎处下方、栓线插入的上方剪断右颈外动脉，撤离颈内动脉处的微血管夹，并使栓线插入端在右颈总动脉分叉处。将颈外动脉拉向外下方，使其与颈内动脉处于同一直线。将栓线向颈内动脉方向插入直至感受到阻力，为防止出血将栓线扎紧。撤离微血管夹，缝合切口。
b)根据相关文献介绍方法进行脑梗死体积测量，具体为断头处死大鼠，迅速取出鼠脑，置于冰盐水中10min，取冠状面均匀切成2mm厚脑片，迅速放入1％的TTC溶液中于37℃染色30min，然后用4％多聚甲醛缓冲液固定。24h后用数码相机拍照，输入计算机，用图像处理软件(ADOBE PHOTOSHOP CS6)计算每片脑片的梗死面积(粉红色区为正常脑组织，白色区为梗死区)后积分转换为梗死容积。梗死容积百分比＝每片脑片的梗死区域体积之和/每片脑片的全脑组织体积之和×100％。
c)脑纹状体注射rAAV9病毒：在293T细胞进行病毒包装，qPCR测定其滴度为3.2×10 12-3.5×10 12V.G./ml。使用的针对于HK2的干扰序列为：5′-GCGCAACATTCTCATCGATTT-3′；5′-AAATCGATGAGAATGTTGCGC-3′；乱序对照组shRNA序列为TTCTCCGAACGTG TCACGT。使用脑立体定位仪将上述病毒分别注入大脑纹状体区域，具体坐标为：前囟前方1.0mm，中线右侧3.0mm，硬膜下4.5mm。注射病毒体积为2μL，注射速度为每分钟0.2μL。注射完毕后，微量注射剂停留在注射部位5分钟之后再拔出。
d)组织免疫荧光检测病毒在脑内的分布(eGFP)，己糖激酶2以及Iba-1(小胶质细胞激活的另一个分子标记物)的表达：大鼠脑缺血2小时再灌注24小时后经麻醉取出完整的脑组织，经固定石蜡包埋后切片，脑片厚度大约为4μm。脑片经过脱蜡，复水以及抗原修复后孵育上述抗体，4℃过夜。次日加入荧光标记二抗，37℃孵育1小时。孵育结束后，加入DAPI工作液进行细胞核染色10分钟。处理结束后，以PBST洗涤三次并利用激光共聚焦显微镜进行成像。
e)免疫组织化学检测IL-1β的表达：大鼠脑缺血2小时再灌注24小时后经麻醉取出完整的脑组织，经固定石蜡包埋后切片，脑片厚度大约为4μm。脑片经过脱蜡，复水以及抗原修复后孵育上述抗体，4℃过夜。次日样品经过PBS洗涤后加入HRP连接的二抗，室温孵育15分钟后，加入DAB显色液孵育1分钟。最后，用苏木精进行细胞核染色，之后利用尼康显微镜进行成像。
结果：
注射rAAV9-shHK2和rAAV9-shNC 20天后，称量两组动物体重，经过统计分析后并没有显著差异(两组动物体重分别为273.2±6.9g和269.3±5.0g)。手术前每组挑选三只取出完整脑组织后根据eGFP的表达检测病毒在脑内的分布，9A成像结果显示病毒在大脑内有较好的组织分布。手术后切片经染色显示：与正常对照相比，手术梗死侧己糖激酶2显著上调表达。而随着病毒的扩散，rAAV9-shHK2组的己糖激酶2表达被明显抑制，而注射rAAV9-shNC后对己糖激酶2的表达并没有显著影响(如图9B所示)。TTC染色结果表明(9C和9D所示)，与rAAV9-shNC组大鼠相比，rAAV9-shHK2组大鼠的脑梗死面积有显著减少。同时如9E所示，纹状体区域Iba-1染色显示在rAAV9-shNC组大鼠脑梗死半球，小胶质细胞突起缩起，呈现激活形态；而shHK2组，小胶质细胞呈现正常形态，且Iba-1的表达也有明显下降。如9F所示，对皮层和纹状体区域的Il-1β进行免疫组织化学染色表明：体内己糖激酶2的敲低所介导的神经保护作用是与降低Il-1β的表达相关的；在对照组，Il-1β分布在皮层和纹状体区域，而在rAAV9-shHK2组，Il-1β的表达被显著抑制。

Claims (10)

  1. 一种己糖激酶2特异性抑制剂在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。
  2. 根据权利要求1所述的应用，其中所述己糖激酶2特异性抑制剂为己糖激酶2的抗体或其片段、或干扰己糖激酶2的mRNA的siRNA、shRNA、miRNA或其修饰物。
  3. 根据权利要求2所述的应用，其中所述己糖激酶2的抗体以编码该抗体的氨基酸序列、核苷酸序列、或包含该核苷酸序列或氨基酸序列的表达载体的形式存在。
  4. 根据权利要求3所述的应用，其中所述己糖激酶2抗体以融合蛋白的形式存在于表达载体或宿主细胞中。
  5. 根据权利要求3所述的应用，其中所述抗体的片段为Fab、Fab'、(Fab') 2或Fv。
  6. 根据权利要求2所述的应用，其中所述siRNA、shRNA、miRNA或其修饰物存在于特定的载体中。
  7. 根据权利要求1所述的应用，其中所述急性中枢神经系统损伤疾病选自急性脊柱损伤、脑外伤、视网膜损伤、缺氧性脑损伤、急性缺血性脑损伤、缺血性脑卒中、缺氧性脑卒中、新生儿缺氧缺血性脑病、中毒性脑病、急性脑梗塞、腔隙性脑梗死、短暂性脑缺血发作、重型颅脑损伤、脑脊髓手术和脑脊髓放疗等导致的脑、脊髓神经损伤。
  8. 一种包含己糖激酶2特异性抑制剂的组合物在制备预防和治疗急性中枢神经系统损伤疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其中所述己糖激酶2特异性抑制剂为己糖激酶2的抗体或其片段、或干扰己糖激酶2的mRNA的siRNA、shRNA、miRNA或其修饰物。
  10. 根据权利要求8所述的应用，其中所述包含己糖激酶2特异性抑制剂的组合物还包含以下化合物中的一种或多种：2-脱氧葡萄糖、氯尼达明、3-溴丙酮酸、6-磷酸葡萄糖、伊马替尼、5-硫代葡萄糖和茉莉酮酸甲酯。
PCT/CN2018/075401 2017-02-23 2018-02-06 己糖激酶2特异性抑制剂在急性中枢神经系统损伤疾病中的应用 WO2018153244A1 (zh)

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