WO2023217249A1 - HTR2b激活剂在改善脑缺血再灌注损伤中的应用 - Google Patents

HTR2b激活剂在改善脑缺血再灌注损伤中的应用 Download PDF

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WO2023217249A1
WO2023217249A1 PCT/CN2023/093663 CN2023093663W WO2023217249A1 WO 2023217249 A1 WO2023217249 A1 WO 2023217249A1 CN 2023093663 W CN2023093663 W CN 2023093663W WO 2023217249 A1 WO2023217249 A1 WO 2023217249A1
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reperfusion injury
cerebral ischemia
htr2b
activator
macrophages
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PCT/CN2023/093663
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English (en)
French (fr)
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李佩盈
张月蔓
俞卫锋
黄婷婷
李妍
谢婉晴
陈晨
陈惟洁
郭昀璐
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上海交通大学医学院附属仁济医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of biomedicine, and specifically to the application of an HTR2b activator in improving cerebral ischemia-reperfusion injury.
  • ischemic stroke has ranked among the top three diseases burdening the world's population, and stroke and ischemic heart disease were the leading causes of death and disability nationwide in 2017. main reason.
  • Acute ischemic stroke is a common type of stroke, accounting for 60% to 80% of all strokes.
  • rt-PA human tissue plasminogen activator
  • intravascular mechanical thrombectomy treatment With the development of the above vascular opening treatments, the damage caused by cerebral ischemia-reperfusion cannot be ignored.
  • FDA-approved drugs or non-drug neuroprotective therapies that can be used to treat reperfusion injury related to blood flow reconstruction or nerve damage outside the time window of thrombolysis and thrombectomy.
  • the neuroinflammatory response after cerebral ischemia-reperfusion injury has gradually become a major focus of research in recent years.
  • the research on the neuroinflammatory response caused by peripheral immune cell infiltration after reperfusion has made great progress in recent years. breakthrough.
  • a large number of immune cells will infiltrate into the cerebral ischemia area and be activated, including blood-derived monocytes/macrophages, neutrophils and lymphocytes, which play a key role in the occurrence and development of neuroinflammation. effect.
  • the multiple roles played by macrophages have attracted particular attention in recent years.
  • monocytes/macrophages were recruited from the bone marrow and migrated to the brain through the blood and began to attack adjacent and dead brain tissues; while in the acute phase after cerebral ischemia, In the subacute phase after perfusion injury, monocytes/macrophages infiltrating into the brain effectively eliminate apoptotic neurons in the infarct area. Therefore, monocytes/macrophages undergo relatively complex changes after cerebral ischemia-reperfusion injury, and play a very important role in the prognosis of cerebral ischemia-reperfusion injury.
  • peripheral/mononuclear macrophages infiltrating into the brain affect cerebral ischemic injury remains an unknown issue. Due to previous technical limitations, there are limited means to distinguish between centrally located microglia and peripheral infiltrating mononuclear macrophages after cerebral ischemia-reperfusion injury. Therefore, for the mononuclear macrophages infiltrating into the brain after cerebral ischemia-reperfusion injury, The complexity of how cells influence the outcome of brain injury is poorly understood.
  • the object of the present invention is to provide the use of HTR2B activators in preparing drugs for improving cerebral ischemia-reperfusion injury, as well as a method for treating and/or preventing cerebral ischemia-reperfusion injury.
  • a first aspect of the present invention provides the use of an HTR2b receptor or its activator for preparing a preparation or composition for:
  • the activator includes small molecule compounds, nucleic acids, proteins or combinations thereof.
  • the activator is selected from the following group: BW723C86, 5-HT, or a combination thereof.
  • the activator is BW723C86.
  • the monocyte macrophage specifically expresses HTR2b receptor highly.
  • the mononuclear macrophages are mononuclear macrophages that infiltrate into the brain from the periphery.
  • reducing cerebral ischemia-reperfusion injury includes: reducing cerebral infarction area, and/or reducing blood-brain barrier (BBB) damage.
  • BBB blood-brain barrier
  • a second aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising:
  • the pharmaceutical composition is used to treat and/or prevent cerebral ischemia-reperfusion injury.
  • the activator includes small molecule compounds, nucleic acids, proteins or combinations thereof.
  • the HTR2b receptor activator is selected from the following group: BW723C86, 5-HT, or a combination thereof.
  • the HTR2b receptor activator is BW723C86, and its effective dose for treating and/or preventing cerebral ischemia-reperfusion injury is 1-10 mg/kg (body weight), preferably 1-5 mg /kg (body weight), optimally 2-4mg/kg (body weight).
  • the other drugs for treating and/or preventing cerebral ischemia-reperfusion injury include: anti-ICAM-1 antibody, E-selectin, minocycline, fingolimod or a combination thereof.
  • the pharmaceutical composition is prepared as a liquid preparation or a freeze-dried preparation.
  • the pharmaceutical composition is prepared as an injection.
  • the administration route of the pharmaceutical preparation includes: intravenous injection, intravenous infusion, and intracranial injection.
  • a third aspect of the present invention provides a pharmaceutical kit, which contains:
  • a first formulation in a first container comprising (a) an HTR2b receptor activator as an active ingredient, and (b) a pharmaceutically acceptable carrier;
  • kit (III) Instructions indicating that the kit is used to treat and/or prevent cerebral ischemia-reperfusion injury.
  • first preparation and the second preparation are independent.
  • the first preparation is a freeze-dried preparation or a liquid preparation, preferably a liquid preparation.
  • the first preparation is an injection.
  • the first formulation is administered before or after the second formulation, or simultaneously with the second formulation.
  • the activator includes small molecule compounds, nucleic acids, proteins or combinations thereof.
  • the HTR2b receptor activator is selected from the following group: BW723C86, 5-HT, or a combination thereof, preferably BW723C86.
  • the other drugs for treating and/or preventing cerebral ischemia-reperfusion injury include: anti-ICAM-1 antibody, E-selectin, minocycline, fingolimod or a combination thereof.
  • a fourth aspect of the present invention provides a method for enhancing the phagocytic capacity of mononuclear macrophages in vitro, which method includes: administering an HTR2b receptor activator to the mononuclear macrophages.
  • the activator includes small molecule compounds, nucleic acids, proteins or combinations thereof.
  • the HTR2b receptor activator is selected from the following group: BW723C86, 5-HT, or a combination thereof.
  • the HTR2b receptor activator is BW723C86.
  • the mononuclear macrophages are primary bone marrow-derived macrophages.
  • the method is non-diagnostic and non-therapeutic.
  • a fifth aspect of the present invention provides a method for treating and/or preventing cerebral ischemia-reperfusion injury, the method comprising the steps of: administering an HTR2b receptor activator to a subject in need, or the second method of the present invention.
  • the pharmaceutical composition described in the aspect is described in the aspect.
  • the activator includes small molecule compounds, nucleic acids, proteins or combinations thereof.
  • the HTR2b receptor activator is selected from the following group: BW723C86, 5-HT, or a combination thereof.
  • the HTR2b receptor activator is BW723C86.
  • the subject in need thereof includes humans or non-human mammals.
  • the cerebral ischemia-reperfusion injury is cerebral ischemia-reperfusion injury caused by ischemic stroke.
  • Figure 1 shows the specific enhancement of neurotransmitter pathways in peripheral infiltration of mononuclear macrophages into the brain after MCAO.
  • A t-SNE plot used to identify monocyte macrophages from different models and tissue sources, with each point representing one cell.
  • B t-SNE plot was used to identify 13 different clusters of monocyte macrophages, among which clusters 3 and 4 are monocyte macrophages entering the brain from the periphery.
  • C Heat map shows the results of KEGG pathway analysis of 13 clusters of monocyte macrophages, in which the blue box indicates that the 5-HT and GABA receptor pathways of macrophages entering the brain (clusters 3, 4) are significantly enriched. set.
  • Figure 2 shows specific context-dependent upregulation of HTR2B receptors in mononuclear macrophages peripherally infiltrating into the brain.
  • A Expression of various neurotransmitter receptor subtypes in different clusters (0-12) of monocyte macrophages.
  • Figure 3 shows that HTR2B receptor-specific agonists can regulate the phagocytosis function of mononuclear macrophages and effectively improve the cerebral infarction area and BBB damage in mice 3 days after MCAO.
  • A Representative MAP2 and IgG staining of cerebral infarction in mice 3 days after stroke,
  • B-C quantification of infarct volume and IgG+ volume, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 4 shows (A) DEGs of primary bone marrow-derived macrophages after administration of HTR2B receptor-specific activator (BW723C86) and PBS.
  • BC HTR2B receptor-specific activator
  • BW723C86 HTR2B receptor-specific activator
  • FIG. 4 shows (A) DEGs of primary bone marrow-derived macrophages after administration of HTR2B receptor-specific activator (BW723C86) and PBS.
  • BC HTR2B receptor-specific activator
  • BW723C86 HTR2B receptor-specific activator
  • SB204741 inhibitor
  • the inventor unexpectedly discovered for the first time that the neurotransmitter receptor HTR2b plays an important role in regulating the phagocytic function of macrophages and affecting ischemia-reperfusion brain injury, and proposed a method for treating cerebral ischemia-reperfusion.
  • a new approach to perfusion injury Through single-cell RNA sequencing and immunofluorescence experiments, the inventors determined the specific environment-dependent upregulation of HTR2b receptors on monocytes/macrophages peripherally infiltrating into the brain after cerebral ischemia-reperfusion injury, and further verified that the HTR2b receptor was upregulated in the brain after cerebral ischemia-reperfusion injury.
  • HTR2b receptor-specific agonists such as BW723C86
  • BW723C86 BW723C86
  • in vitro experiments have also proven that HTR2b receptor-specific agonists can significantly enhance the phagocytosis function of monocytes/macrophages.
  • 5-HT receptors also known as serotonin receptors, are a group of G-protein-coupled receptors and ligand ion channels found in the nervous system, which can be divided into seven subfamilies, 5-HT1-7.
  • the 5-HT2 receptor family includes three subtypes: HTR2a, HTR2b and HTR2c.
  • HTR2b is a specific receptor for 5-hydroxytryptamine (5-HT), which can regulate mood, behavior and cognition.
  • Literature suggests that HTR2b but not HTR2a is up-regulated in an age-dependent manner after cerebral ischemia-reperfusion injury. Previous studies have found that HTR2b receptor activation may be involved in human macrophage polarization and help maintain an anti-inflammatory state. Hajer El Oussini et al. also found that the presence of HTR2b receptor limits the degeneration of spinal cord mononuclear phagocytes and slows the progression of ALS disease.
  • the present invention provides the use of an HTR2b receptor activator, wherein the activator includes (but is not limited to) small molecule compounds, nucleic acids (such as DNA, RNA), and proteins (such as enzymes, antibodies, etc.).
  • the activator includes (but is not limited to) small molecule compounds, nucleic acids (such as DNA, RNA), and proteins (such as enzymes, antibodies, etc.).
  • the 5-HT2B receptor is a G protein-coupled receptor for the endogenous neurotransmitter serotonin (5-HT).
  • BW723C86 (hydrochloride) is a tryptamine analog with a molecular formula of C 16 H 19 ClN 2 OS and a molecular weight of 322.85. It is a highly selective 5-HT2B receptor-specific agonist, and is different from 5-HT2A and 5 -HT2C, 5-HT1A and 5-HT1B receptor binding is weak.
  • BW723C86 has a non-amphetamine-like structure. Amphetamine molecules, such as fenfluramine, are anorexigenic, and dietary restriction is associated with stroke prognosis.
  • the present invention provides a pharmaceutical composition, which can be used to: (i) up-regulate the phagocytic function of monocyte macrophages; and/or (ii) prevent and/or reduce cerebral ischemia-reperfusion injury, wherein said "reducing "Cerebral ischemia-reperfusion injury” includes reducing cerebral infarct size and/or alleviating blood-brain barrier (BBB) damage.
  • a pharmaceutical composition which can be used to: (i) up-regulate the phagocytic function of monocyte macrophages; and/or (ii) prevent and/or reduce cerebral ischemia-reperfusion injury, wherein said "reducing "Cerebral ischemia-reperfusion injury” includes reducing cerebral infarct size and/or alleviating blood-brain barrier (BBB) damage.
  • BBB blood-brain barrier
  • the pharmaceutical composition of the present invention includes: (a) an HTR2b receptor activator, and a pharmaceutically acceptable carrier; and (b) other drugs for treating and/or preventing cerebral ischemia-reperfusion injury, and a pharmaceutically acceptable carrier. .
  • the HTR2b receptor activator of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • pharmaceutically acceptable carrier refers to pharmaceutical carriers that do not themselves require the active ingredient and are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, and buffers. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc.
  • the vector may also contain cell transfection reagents.
  • the term "effective amount” or “effective dosage” refers to an amount that produces a function or activity in humans and/or animals and/or cells and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use by humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance that has a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, pharmaceutical preparations should match the mode of administration.
  • the pharmaceutical preparations of the present invention can be prepared in the form of injections, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the active ingredients are administered in amounts that are therapeutically effective.
  • the pharmaceutical combination of the present invention can also be made into a sustained-release preparation.
  • a pharmaceutical formulation When a pharmaceutical formulation is used, a safe and effective amount of a combination of active ingredients, including an HTR2b receptor activator, is administered to the mammal.
  • a combination of active ingredients including an HTR2b receptor activator
  • the effective amount of the active ingredients of the present invention may vary depending on the mode of administration and the severity of the tumor.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters such as Bioavailability, metabolism, half-life, etc.; tumor severity, patient's weight, patient's immune status, route of administration, etc.
  • the "effective amount" of HTR2b receptor activator BW723C86 is a plasma concentration of 200ng/g (a one-time injection of 3mg/kg (body weight) can last for 8 hours).
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited. Representative examples include (but are not limited to) intravenous injection, intravenous infusion, intracranial injection, subcutaneous injection, intramuscular injection, etc.
  • the invention provides a medicine box, which includes:
  • a first formulation in a first container comprising (a) an HTR2b receptor activator as an active ingredient, and (b) a pharmaceutically acceptable carrier;
  • kit (III) Instructions indicating that the kit is used to treat and/or prevent cerebral ischemia-reperfusion injury.
  • the first preparation includes (but is not limited to): freeze-dried preparations and liquid preparations (such as injections).
  • the second preparation includes (but is not limited to): solid preparations (such as tablets, capsules, powders) and liquid preparations.
  • the kit contains one or more (eg, at least two) unit dosage forms containing an HTR2b receptor activator and one or more (eg, at least two) unit dosage forms containing an agent for treating and/or preventing cerebral ischemia-reperfusion injury.
  • Unit dosage forms of other drugs preferably 4-10 units each.
  • unit dosage form refers to a composition prepared into the dosage form required for a single administration for convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, sustained release dosage forms, etc. agent.
  • the instructions provided by the present invention may include the following description: the method of using the kit is to simultaneously use a unit dosage form containing an HTR2b receptor activator and a unit dosage form containing other drugs for treating and/or preventing cerebral ischemia-reperfusion injury.
  • the medicine kit is particularly suitable for cerebral ischemia-reperfusion injury caused by ischemic stroke.
  • the pharmaceutical kit provided by the present invention is prepared by the following steps: placing a preparation containing an HTR2b receptor activator, a preparation of other drugs for treating and/or preventing cerebral ischemia-reperfusion injury, and instructions together to form a pharmaceutical kit.
  • the preparation containing HTR2b receptor activator preferably contains a unit dosage form of HTR2b receptor activator
  • the preparation containing other drugs for treating and/or preventing cerebral ischemia-reperfusion injury preferably contains a unit dosage form of other drugs for treating and/or preventing cerebral ischemia-reperfusion injury.
  • the step preferably places at least one unit dosage form containing an HTR2b receptor activator and at least one unit dosage form containing other drugs for treating and/or preventing cerebral ischemia-reperfusion injury together with instructions to form a kit.
  • the present invention also provides a method for enhancing the phagocytic capacity of mononuclear macrophages in vitro, which method includes: administering an HTR2b receptor activator to the mononuclear macrophages.
  • the HTR2b receptor activator BW723C86 is administered to the primary monocyte macrophage BMDM (Bone Marrow-Derived Macrophage) cultured in vitro, thereby enhancing the phagocytic ability of the monocyte macrophage BMDM.
  • the present invention also provides a method for treating and/or preventing cerebral ischemia-reperfusion injury, the method comprising the steps of: administering an HTR2b receptor activator to a subject in need, or the method described in the second aspect of the present invention.
  • Pharmaceutical compositions comprising: administering an HTR2b receptor activator to a subject in need, or the method described in the second aspect of the present invention.
  • Pharmaceutical compositions by administering the HTR2b receptor activator BW723C86 to the test animal (ischemic stroke model mouse), the phagocytic ability of mononuclear macrophages infiltrating into the brain from the periphery is enhanced, thereby Improved cerebral infarction area and BBB damage in mice.
  • mice were first anesthetized with 2% isoflurane in 30% O 2 /70% N 2 mixture in air. After complete anesthesia, a skin incision was made on the mouse's neck to expose the left common carotid artery and its branches. The common carotid artery was temporarily ligated, and then the external carotid artery was cut off. The suture plug was inserted from the external carotid artery into the internal carotid artery through the external carotid artery stump, and MCAO was induced by intraluminal occlusion of the left middle cerebral artery for 1 hour. Sham-operated animals received arterial anesthesia and surgical exposure but did not undergo middle cerebral artery occlusion. During the surgery, use a heating pad to maintain the mouse's body temperature at 37 ⁇ 0.5°C.
  • Cell lysis buffer is added to allow polyadenylated RNA molecules to hybridize to the beads. Collect beads into individual tubes for reverse transcription.
  • each BD Rhapsody system is used to obtain transcriptomic information from single cells. Single cell capture is achieved by randomly distributing a single cell suspension in >200,000 microwells using a limiting dilution method. Beads with oligonucleotide codes were added to saturation to allow beads to pair with cells in the wells.
  • the cDNA molecule is tagged at the 5' end (i.e., the 3' end of the mRNA transcript) with a unique molecular identifier (UMI) and a cellular marker indicating the cell of its origin.
  • UMI unique molecular identifier
  • Cerebral infarct size was measured using microtubule-associated protein 2 (MAP-2) staining, and the animals were sacrificed and perfused transcardially with 0.9% saline and 4% paraformaldehyde in PBS. Free-floating sections of different sections were prepared from fixed and dehydrated brains and then stained with MAP-2 antibody (1:500, abcam). Infarct volume was determined by NIH Image J (1.52a) analysis by an observer unknown to experimental group assignment.
  • MAP-2 microtubule-associated protein 2
  • mice were anesthetized and sacrificed by dislocation.
  • the mice were placed in a beaker containing a sufficient amount of 75% ethanol and soaked and sterilized. After 5 minutes, soak the soaked animals with paper to absorb excess alcohol.
  • Use scissors to cut a small opening on the mouse's back tear the skin directly to the mouse's calf joints with your hands, and remove the mouse's foot joints and skin (this process is slightly rough and bloody, but very efficient).
  • Use scissors to remove the hind limb along the greater trochanter at the root of the mouse's thigh remove the muscle tissue, place it in a petri dish containing 75% ethanol and soak it for 5 minutes, and replace it with a new one.
  • the petri dish with 75% ethanol was moved to a clean bench.
  • BMDM cells were plated on 1.5-mm coverslips at a density of 2 ⁇ 10 cells/well for 24 h. Then, fluorescently labeled latex beads were added at a concentration of 5 ⁇ l/ml for 2 h at 37°C. Cells were washed three times with PBS to remove unphagocytosed beads and fixed with 4% paraformaldehyde. Next, phagocytosis of beads by microglia was observed under a fluorescence inverted microscope. Cells with phagocytic activity were observed using fluorescence microscopy ( ⁇ 40 and ⁇ 100 magnification). All experiments were repeated three times. Microglia in the negative control group were pretreated with cytochalasin D (10 ⁇ M) for 30 min.
  • Example 1 Single-cell RNA sequencing reveals peripheral infiltration of monocyte-macrophage neurotransmitter pathways into the brain Enhanced specificity
  • the macrophage marker-Ms4a7 is used to specifically mark monocytes and macrophages and monocytes/macrophages in the bone marrow for in-depth analysis.
  • Example 2 Specific context-dependent upregulation of HTR2b receptor in monocyte macrophages peripherally infiltrating into the brain
  • HTR2b receptors are specifically highly expressed in peripheral cells entering the brain.
  • macrophages clusters 3, 4 in the brain ( Figure 2A), suggesting that peripheral monocytes/macrophages with specific high expression of HTR2b receptors may play a very important role in cerebral ischemia-reperfusion injury.
  • Example 3 HTR2b receptor specific agonist effectively improves cerebral infarction area and BBB damage in mice 3 days after MCAO
  • HTR2b receptor-specific agonist BW723C86 2 hours after MCAO and found that it can effectively improve post-MCAO
  • BW723C86 HTR2b receptor-specific agonist
  • Example 4 HTR2b receptor-specific agonists can regulate the phagocytic function of monocytes and macrophages
  • HTR2b receptor against ischemic cerebral ischemia-reperfusion injury works by changing the phagocytic function of mononuclear macrophages
  • primary bone marrow-derived macrophages were given dose-dependent HTR2b receptor specificity.
  • the agonist could significantly enhance the phagocytosis function of primary macrophages BMDM ( Figure 4D).
  • HTR2b receptor regulates the outcome of cerebral ischemia-reperfusion injury by affecting the phagocytic function of macrophages.

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Abstract

一种HTR2b受体或其激活剂用于制备制剂或组合物的用途，制剂或组合物用于：(i)上调单核巨噬细胞的吞噬功能；和/或(ii)预防和/或减轻脑缺血再灌注损伤。激活剂选自下组：BW723C86、5-HT、或其组合。

Description

HTR2b激活剂在改善脑缺血再灌注损伤中的应用 技术领域
本发明涉及生物医药领域，具体地涉及一种HTR2b激活剂在改善脑缺血再灌注损伤中的应用。
背景技术
最近的全球疾病负担研究表明，缺血性脑卒中(Ischemic Stroke)已跃居全世界人口疾病负担的前三位，且卒中和缺血性心脏病是2017年我国全国范围内死亡和残疾的最主要原因。急性缺血性脑卒中是常见的脑卒中类型，占全部脑卒中的60％～80％。然而根据2018年AHA新发布的脑卒中治疗指南,除了重组人组织型纤溶酶原激活剂(rt-PA)溶栓以及血管内机械性取栓治疗。随着以上血管开通治疗的发展，脑缺血再灌注的损伤显得越来越不容忽视。然而目前尚无经FDA批准的药物或非药物的神经保护疗法可以用来治疗血流重建相关的再灌注损伤或溶栓取栓时间窗以外的神经损伤。
脑缺血再灌注损伤后的神经炎症反应逐渐在近几年的研究中成为了一大焦点，尤其是再灌注后外周免疫细胞浸润导致的神经炎症反应研究更是在近几年有了很大的突破。脑缺血再灌注后大量的免疫细胞会浸润至脑缺血区域并激活，包括血液来源的单核/巨噬细胞、中性粒细胞及淋巴细胞，在神经炎症的发生和发展中起着关键作用。在这些细胞中，巨噬细胞所起的多重作用在近几年来格外引人关注。Ralf Stumm等人利用Cxcr4-GFP小鼠观察在中风后急性期，从骨髓中募集经血液迁移至脑的大量单核/巨噬细胞开始攻击邻近的和死亡的大脑组织；而在脑缺血再灌注损伤后亚急性期，浸润入脑的单核/巨噬细胞有效的清除了梗死区的凋亡神经元。因此，单核/巨噬细胞在脑缺血再灌注损伤后发生了较为复杂的变化，并对脑缺血再灌注损伤的预后有十分重要的作用。然而目前，外周浸润入脑的/单核巨噬细胞如何影响脑缺血损伤仍是一个未知的问题。而由于既往技术限制，对脑缺血再灌注损伤后中枢固有小胶质细胞和外周浸润的单核巨噬细胞区别手段有限，因此对于脑缺血再灌注损伤后浸润入脑的单核巨噬细胞如何影响脑损伤结局的复杂性了解甚少。
发明内容
本发明的目的在于提供HTR2B激活剂在制备用于改善脑缺血再灌注损伤的药物中的应用，以及一种治疗和/或预防脑缺血再灌注损伤的方法。
本发明的第一方面，提供了一种HTR2b受体或其激活剂的用途，用于制备一制剂或组合物，所述制剂或组合物用于：
(i)上调单核巨噬细胞的吞噬功能；和/或
(ii)预防和/或减轻脑缺血再灌注损伤。
在另一优选例中，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
在另一优选例中，所述激活剂选自下组：BW723C86、5-HT、或其组合。
在另一优选例中，所述激活剂为BW723C86。
在另一优选例中，所述单核巨噬细胞特异性高表达HTR2b受体。
在另一优选例中，所述单核巨噬细胞是由外周浸润入脑的单核巨噬细胞。
在另一优选例中，所述“减轻脑缺血再灌注损伤”包括：减小脑梗死面积，和/或减轻血-脑屏障(BBB)损伤。
本发明的第二方面，提供了一种药物组合物，所述药物组合物包含：
(a)HTR2b受体激活剂，和药学上可接受的载体；以及
(b)治疗和/或预防脑缺血再灌注损伤的其他药物，和药学上可接受的载体。
在另一优选例中，所述药物组合物用于治疗和/或预防脑缺血再灌注损伤。
在另一优选例中，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
在另一优选例中，所述HTR2b受体激活剂选自下组：BW723C86、5-HT、或其组合。
在另一优选例中，所述HTR2b受体激活剂为BW723C86，其用于治疗和/或预防脑缺血再灌注损伤的有效量为1-10mg/kg(体重)，较佳地1-5mg/kg(体重)，最佳地2-4mg/kg(体重)。
在另一优选例中，所述治疗和/或预防脑缺血再灌注损伤的其他药物包括：抗ICAM-1抗体、E-选择素、米诺环素、芬戈莫德或其组合。
在另一优选例中，所述药物组合物被制备为液态制剂或冻干制剂。
在另一优选例中，所述药物组合物被制备为注射剂。
在另一优选例中，所述药物制剂的给药途径包括：静脉注射、静脉输注、颅内注射。
本发明的第三方面，提供了一种药盒，所述药盒包含：
(I)位于第一容器内的第一制剂，所述第一制剂包含(a)HTR2b受体激活剂作为活性成分，和(b)药学上可接受的载体；
(II)位于第二容器内的第二制剂，所述第二制剂包含治疗和/或预防脑缺血再灌注损伤的其他药物作为活性成分；和
(III)说明书，所述说明书注明所述药盒用于治疗和/或预防脑缺血再灌注损伤。
在另一优选例中，所述的第一制剂和第二制剂是各自独立的。
在另一优选例中，所述的第一制剂为冻干制剂或液态制剂，优选为液态制剂。
在另一优选例中，所述的第一制剂为注射剂。
在另一优选例中，所述第一制剂在第二制剂施用之前或之后施用，或与第二制剂同时施用。
在另一优选例中，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
在另一优选例中，所述HTR2b受体激活剂选自下组：BW723C86、5-HT、或其组合，优选BW723C86。
在另一优选例中，所述治疗和/或预防脑缺血再灌注损伤的其他药物包括：抗ICAM-1抗体、E-选择素、米诺环素、芬戈莫德或其组合。
本发明的第四方面，提供了一种体外增强单核巨噬细胞吞噬能力的方法，所述方法包括：向单核巨噬细胞施用HTR2b受体激活剂。
在另一优选例中，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
在另一优选例中，所述HTR2b受体激活剂选自下组：BW723C86、5-HT、或其组合。
在另一优选例中，所述HTR2b受体激活剂为BW723C86。
在另一优选例中，所述单核巨噬细胞为原代骨髓来源巨噬细胞。
在另一优选例中，所述方法为非诊断和非治疗性的。
本发明的第五方面，提供了一种治疗和/或预防脑缺血再灌注损伤的方法，所述方法包括步骤：向有需要的受试者施用HTR2b受体激活剂，或本发明第二方面所述的药物组合物。
在另一优选例中，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
在另一优选例中，所述HTR2b受体激活剂选自下组：BW723C86、5-HT、或其组合。
在另一优选例中，所述HTR2b受体激活剂为BW723C86。
在另一优选例中，所述有需要的受试者包括人类或非人类哺乳动物。
在另一优选例中，所述脑缺血再灌注损伤为缺血性脑卒中导致的脑缺血再灌注损伤。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了MCAO后外周浸润入脑的单核巨噬细胞神经递质通路特异性增强。(A)t-SNE图用于鉴定不同模型和组织来源的单核巨噬细胞，每个点代表一个细胞。(B)t-SNE图用于鉴定单核巨噬细胞分成的13个不同的簇，其中簇3，4是从外周进入脑中的单核巨噬细胞。(C)热图显示单核巨噬细胞13个簇的KEGG通路分析结果，其中蓝色方框表明进入脑中的巨噬细胞(簇3，4)的5-HT和GABA受体通路明显富集。
图2显示了外周浸润入脑的单核巨噬细胞HTR2B受体特异性环境依赖性上调。(A)各类神经递质受体亚型在单核巨噬细胞不同簇(0-12)的表达情况。(B)免疫荧光显示MCAO后3d和Sham小鼠的脑梗死周围HTR2B的表达，比例尺＝50μm。
图3显示了HTR2B受体特异性激动剂可以调节单核巨噬细胞吞噬功能有效改善MCAO后3d小鼠的脑梗死面积以及BBB损伤。(A)为脑卒中后3天小鼠脑梗死的代表性MAP2和IgG染色,(B-C)为梗死体积和IgG+体积的量化，*p≤0.05，**p≤0.01。
图4显示了(A)给与HTR2B受体特异性激活剂(BW723C86)和PBS后的原代骨髓来源巨噬细胞的DEG。(B-C)对A中DEG的KEGG通路和GESA分析。(D)使用原代骨髓来源巨噬细胞和HTR2B受体特异性激动剂(BW723C86)和抑制剂(SB204741)共培养后进行离体吞噬作用结果的免疫荧光结果示意图和吞噬指 数的测定。比例尺＝20μm，N＝6-9/组，*p≤0.05，**p≤0.01。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现神经递质受体HTR2b在调控巨噬细胞吞噬功能，以及影响缺血再灌注脑损伤中的重要作用，提出了一种治疗脑缺血再灌注损伤的新方法。本发明人通过单细胞RNA测序和免疫荧光实验，确定了脑缺血再灌注损伤后外周浸润入脑的单核/巨噬细胞HTR2b受体特异性环境依赖性上调，并进一步验证了向脑缺血再灌注损伤小鼠模型施用HTR2b受体特异性激动剂(如BW723C86)可有效改善小鼠的脑梗死面积以及BBB损伤。此外，体外实验也证明，HTR2b受体特异性激动剂可以显著增强单核/巨噬细胞的吞噬功能。
在此基础上，完成了本发明。
术语
HTR2b受体
5-HT受体，也被称为血清素受体，是一组发现于神经系统的G蛋白偶联受体和配体离子通道，可分为5-HT1-7七个亚科。其中5-HT2受体家族包括可分为HTR2a，HTR2b以及HTR2c三种亚型，其中HTR2b是5-羟色胺(5-HT)的特异性受体，可调节情绪，行为和认知。文献提示脑缺血再灌注损伤后，HTR2b而非HTR2a出现了年龄依赖性上调。既往研究发现HTR2b受体激活可能参与了人巨噬细胞极化，有助于维持抗炎状态。Hajer El Oussini等人也发现HTR2b受体的存在限制了脊髓单核吞噬细胞的变性，并减缓ALS疾病的进展。
HTR2b受体特异性激活剂
本发明提供了一种HTR2b受体激活剂的用途，其中所述激活剂包括(但不限于)小分子化合物、核酸(例如DNA、RNA)、蛋白(例如酶、抗体等)。
5-HT2B受体是一种内源性神经递质血清素(5-HT)的G蛋白偶联受体。BW723C86(hydrochloride)，是一种色胺类似物，分子式C16H19ClN2OS，分子量是322.85，是一种高选择性的5-HT2B受体特异性激动剂，而与5-HT2A、5-HT2C、5-HT1A和5-HT1B受体结合较弱。BW723C86其非苯丙胺类化学物质结构，而苯丙胺分子，如芬氟拉明，是厌食剂的一种，而饮食限制与卒中的预后相关。
本发明的药物组合物
本发明提供了一种药物组合物，它可用于：(i)上调单核巨噬细胞的吞噬功能；和/或(ii)预防和/或减轻脑缺血再灌注损伤，其中所述“减轻脑缺血再灌注损伤”包括减小脑梗死面积，和/或减轻血-脑屏障(BBB)损伤。
本发明药物组合物包括：(a)HTR2b受体激活剂，和药学上可接受的载体；以及(b)治疗和/或预防脑缺血再灌注损伤的其他药物，和药学上可接受的载体。
通常，可将本发明的HTR2b受体激活剂配制于无毒的、惰性的和药学上可接受的载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
术语“药学上可接受的载体”指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物和/或细胞产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、聚山梨酯、乙醇及其组合。通常药物制剂应与给药方式相匹配，本发明的药物制剂可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物组合我还可制成缓释制剂。
使用药物制剂时，是将安全有效量的活性成分组合(包括HTR2b受体激活剂)施用于哺乳动物。
应理解，本发明所述活性成分的有效量，可随给药的模式和肿瘤的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：药代动力学参数例如生 物利用率、代谢、半衰期等；肿瘤的严重程度、患者的体重、患者的免疫状况、给药的途径等。具体地，在本发明的一个优选实施方式中，HTR2b受体激活剂BW723C86的“有效量”为血浆浓度200ng/g(3mg/kg(体重)一次性注射可维持8h)。
本发明所述的药物组合物的给药方式没有特别限制，代表性的例子包括(但并不限于)：静脉注射、静脉输注、颅内注射、皮下注射、肌肉注射等。
本发明的药盒
本发明提供一种药盒，所述药盒包括：
(I)位于第一容器内的第一制剂，所述第一制剂包含(a)HTR2b受体激活剂作为活性成分，和(b)药学上可接受的载体；
(II)位于第二容器内的第二制剂，所述第二制剂包含治疗和/或预防脑缺血再灌注损伤的其他药物作为活性成分；和
(III)说明书，所述说明书注明所述药盒用于治疗和/或预防脑缺血再灌注损伤。
所述的第一制剂包括(但并不限于)：冻干制剂、液态制剂(如注射剂)。
所述的第二制剂包括(但并不限于)：固体剂(如片剂、胶囊、粉剂)、液体制剂。
典型地，药盒中装有一个或多个(如至少两个)含有HTR2b受体激活剂的单元剂型和一个或多个(如至少两个)含有治疗和/或预防脑缺血再灌注损伤的其他药物的单元剂型；较佳地各为4-10个。
如本文所用，术语“单元剂型”是指为了施用方便，将组合物制备成单次施用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
本发明提供的说明书中可以有如下描述：所述药盒的使用方法是同时使用含有HTR2b受体激活剂的单元剂型和含有治疗和/或预防脑缺血再灌注损伤的其他药物的单元剂型。所述药盒特别适用于由缺血性脑卒中所引起的脑缺血再灌注损伤。
本发明提供的药盒通过下述步骤制备得到：将含有HTR2b受体激活剂的制剂和治疗和/或预防脑缺血再灌注损伤的其他药物的制剂，以及说明书一起放置，形成药盒。
所述的含有HTR2b受体激活剂的制剂优选含有HTR2b受体激活剂的单元剂型， 所述含有治疗和/或预防脑缺血再灌注损伤的其他药物的制剂优选含有治疗和/或预防脑缺血再灌注损伤的其他药物的单元剂型。
所述步骤优选将至少一个含有HTR2b受体激活剂的单元剂型和至少一个含有治疗和/或预防脑缺血再灌注损伤的其他药物的单元剂型，以及说明书一起放置，形成药盒。
本发明的方法
本发明还提供了一种体外增强单核巨噬细胞吞噬能力的方法，所述方法包括：向单核巨噬细胞施用HTR2b受体激活剂。在本发明的一个优选实施方式中，通过向体外培养的原代单核巨噬细胞BMDM(Bone Marrow-Derived Macrophage)施用HTR2b受体激活剂BW723C86，从而增强单核巨噬细胞BMDM的吞噬能力。
本发明还提供了一种治疗和/或预防脑缺血再灌注损伤的方法，所述方法包括步骤：向有需要的受试者施用HTR2b受体激活剂，或本发明第二方面所述的药物组合物。在本发明的一个优选实施方式中，通过向受试动物(缺血脑卒中模型小鼠)施用HTR2b受体激活剂BW723C86，增强了由外周浸润入脑的单核巨噬细胞的吞噬能力，从而改善小鼠的脑梗死面积以及BBB损伤。
本发明的有益效果包括：
1)揭示了脑缺血再灌注损伤后神经系统和外周浸润的单核巨噬细胞相互作用的新机制，揭示了HTR2b受体调控单核巨噬细胞的吞噬功能的新机制；
2)提出脑缺血再灌注损伤后早期特异性激活HTR2b受体的新策略(给予BW723C86)，为脑缺血再灌注损伤的免疫治疗提供新思路，提高患者的生存质量，减轻家庭和社会的负担。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条例，例如J.Sambrook等人，分子克隆实验指南(第四版)(科学出版社有限责任公司，2017)中所述的条件，或按照产品制造商提供的产品说明书中所述条件。实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。
实验方法
1.缺血性脑卒中模型的建立
对C57BL/6背景下的8至10周龄雄性野生型小鼠进行MCAO造模。简而言之，首先用30％O2/70％N2混合空气中混入2％异氟醚麻醉小鼠。麻醉完全之后，在小鼠颈部做一个皮肤切口，暴露左侧颈总动脉及其分支，并暂时结扎颈总动脉，之后截断颈外动脉。并通过颈外动脉残端将线栓自颈外动脉插入颈内动脉，通过腔内阻塞左侧大脑中动脉1小时来诱导MCAO。假手术动物接受了动脉麻醉和手术暴露，但没有进行大脑中动脉闭塞。在手术过程中，使用加热垫将小鼠体温保持在37±0.5℃。
2.单细胞RNA-seq实验
加入细胞裂解缓冲液以使聚腺苷酸化的RNA分子与微珠杂交。将珠子收集到单个管中以进行逆转录。cDNA合成后，每个BD Rhapsody系统均用于获得单细胞的转录组信息。使用有限的稀释方法，通过将单细胞悬液随机分布在>200,000个微孔中，从而实现单细胞捕获。加入具有寡核苷酸码的珠子至饱和，以使珠子与微孔中的细胞配对。cDNA分子在5'端(即mRNA转录物的3'端)被标记，具有唯一的分子标识符(UMI)和指示其来源细胞的细胞标记。使用BD Rhapsody单细胞全转录组扩增工作流程制备了完整的转录组文库。简而言之，合成了第二链cDNA，然后连接WTA衔接子以进行通用扩增。18个PCR循环用于扩增衔接子连接的cDNA产物。使用随机引物PCR制备用于整个转录组扩增产物的测序文库，以富集与细胞标记和UMI连接的转录本的3'端。使用Bioanalyzer2200上的高灵敏度DNA芯片(安捷伦)和Qubit高灵敏度DNA分析(Thermo Fisher Scientific)对测序文库进行定量。通过HiSeq Xten(Illumina，圣地亚哥，加利福尼亚)以150bp的配对末端测序对每个样品的文库进行测序。
3.梗塞体积的测量
利用微管相关蛋白2(MAP-2)染色测量脑梗死面积，处死动物并用0.9％生理盐水和4％多聚甲醛的PBS经心灌注。从固定和脱水的大脑中制备不同断面的自由浮动切片，然后用MAP-2抗体(1:500，abcam)染色。梗死体积由对实验组分配未知的观察者通过NIH Image J(1.52a)分析确定。
4.原代巨噬细胞BMDM(Bone Marrow-Derived Macrophage)的分离和培养
1)小鼠取腿骨
小鼠麻醉后脱臼处死，将小鼠放置在盛有足量75％乙醇的烧杯中浸泡消毒 5min，将浸泡好的动物用纸吸去多余的酒精。用剪刀在小鼠背部剪开一小口，用手直接撕开皮肤至小鼠小腿关节处，去除小鼠足关节以及皮肤(此过程略显粗暴血腥，但效率很高)。用剪刀沿着小鼠大腿根部大转子将后肢拆(拆不是折哦，千万不要将腿骨折断了)下来，去掉肌肉组织后放置在含有75％乙醇的培养皿内浸泡5min，更换新的75％乙醇的培养皿移入超净台中。
2)BMDM细胞提取和诱导
将乙醇浸泡的腿骨移入冷的PBS浸泡，洗去胫骨、股骨表面的乙醇，此过程可重复3次。将清洗好的股骨、胫骨分开，并用剪刀分别将股骨、胫骨两端剪断，使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出，反复吹洗3次，直至腿骨内看不到明显的红色为止。用5mL移液枪将含有骨髓细胞的培养基反复吹打，使细胞团块分散，然后使用70μm细胞滤器将细胞过筛，转移至15mL离心管内，1500rpm/min离心5min，弃上清，加入红细胞裂解液重悬静置5min后1500rpm/min离心5min，弃上清用冷的配置好的骨髓巨噬细胞诱导培养基重悬，铺板。细胞培养期间不更换培养基，培养第三天后更换一半骨髓巨噬细胞诱导培养基，第五天更换全部培养基，第七天即可用于后续实验。
5.吞噬实验
将BMDM细胞以2×104个细胞/孔的密度铺在1.5-mm2盖玻片上24小时。然后，在37℃下以5μl/ml的浓度添加荧光标记的乳胶珠2小时。将细胞用PBS洗涤3次以去除未吞噬的珠子并用4％多聚甲醛固定。接下来，在荧光倒置显微镜下观察小胶质细胞对珠粒的吞噬作用。使用荧光显微镜(×40和×100放大倍数)观察具有吞噬活性的细胞。所有实验重复3次。阴性对照组小胶质细胞用细胞松弛素D(10μM)预处理30min。
6.统计学方法
所有统计数据均使用GraphPad Prism v.6或相应R包的已实施统计测试进行。在应用任何参数分析之前，测试了分布的正态性和方差的相等性。双尾学生t检验用于两组之间的成对比较。其余数据酌情使用单向或双向ANOVA进行分析。使用Bonferroni的事后检验进行多重比较程序以确定特定的组间差异。结果表示为平均值±标准差。使用Pearson相关分析进行相关分析。P≤0.05被认为具有统计学意义。
实施例1单细胞RNA测序显示外周浸润入脑的单核巨噬细胞神经递质通路 特异性增强
为了探究脑缺血再灌注损伤后从外周浸润入脑的单核/巨噬细胞的复杂改变，对建立大脑中动脉阻塞模型(MCAO)后3天小鼠的脑梗死组织进行单细胞测序，并采用RacID 3的方法，使用巨噬细胞标记物-Ms4a7，特异性标记处单核巨噬细胞和骨髓中的单核/巨噬细胞进行深入分析。
结果显示，相对于骨髓中的单核/巨噬细胞，脑缺血再灌注损伤后外周浸润入脑的单核/巨噬细胞的神经递质受体通路出现明显特异性富集，尤其是5-HT和GABA受体通路(图1)。
实施例2外周浸润入脑的单核巨噬细胞HTR2b受体特异性环境依赖性上调
在发现进入脑中的单核巨噬细胞的神经递质受体通路特异性富集的基础上，分析了各类神经递质受体亚型，发现HTR2b受体特异性高表达于外周进入脑中的巨噬细胞(簇3，4)(图2A)，提示HTR2b受体特异性高表达的外周单核/巨噬细胞可能对于脑缺血再灌注损伤起到十分重要的影响。
为了在蛋白水平验证单细胞测序中所观察到的HTR2b受体表达的改变，通过免疫荧光共标的方法，验证了其在MCAO后3d小鼠脑中F4/80+的巨噬细胞中的表达明显增加(图2B)。
以上实验结果提示脑缺血再灌注损伤后，由外周浸润入脑的单核巨噬细胞的神经递质受体HTR2b表达显著上调。
实施例3 HTR2b受体特异性激动剂有效改善MCAO后3d小鼠的脑梗死面积以及BBB损伤
为了探究浸润入脑的单核巨噬细胞的HTR2b受体对脑缺血再灌注损伤的影响，通过在MCAO后2小时给予HTR2b受体特异性激动剂(BW723C86)，发现其可以有效改善MCAO后3天小鼠的脑梗死面积以及BBB损伤(图3)，表明单核巨噬细胞的HTR2b受体的上调可能是脑缺血再灌注损伤以后，机体产生的自发性的保护机制，可减轻脑缺血再灌注的损伤。
实施例4 HTR2b受体特异性激动剂可以调节单核巨噬细胞吞噬功能
为了明确HTR2b受体对缺血性脑缺血再灌注损伤的保护机制，检测了给与HTR2b受体特异性激活剂(BW723C86)后的原代骨髓来源巨噬细胞的转录组改 变，KEGG和GESA分析都发现了给与BW723C86之后吞噬通路明显富集(图4A-C)。
为了明确HTR2b受体对缺血性脑缺血再灌注损伤的保护机制是否通过改变单核巨噬细胞的吞噬功能起效，给与原代骨髓来源巨噬细胞剂量依赖性的HTR2b受体特异性激动剂(BW723C86)和抑制剂(SB204741)进行共培养后，激动剂可以显著增强原代巨噬细胞BMDM的吞噬功能(图4D)。
以上结果表明，HTR2b受体是通过影响巨噬细胞的吞噬功能进而调节脑缺血再灌注损伤的结局。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种HTR2b受体或其激活剂的用途，其特征在于，用于制备一制剂或组合物，所述制剂或组合物用于：
    (i)上调单核巨噬细胞的吞噬功能；和/或
    (ii)预防和/或减轻脑缺血再灌注损伤。
  2. 如权利要求1所述的用途，其特征在于，所述激活剂包括小分子化合物、核酸、蛋白或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述激活剂选自下组：BW723C86、5-HT、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述单核巨噬细胞是由外周浸润入脑的单核巨噬细胞。
  5. 如权利要求1所述的用途，其特征在于，所述“减轻脑缺血再灌注损伤”包括：减小脑梗死面积，和/或减轻血-脑屏障(BBB)损伤。
  6. 一种药物组合物，其特征在于，所述药物组合物包含：
    (a)HTR2b受体激活剂，和药学上可接受的载体；以及
    (b)治疗和/或预防脑缺血再灌注损伤的其他药物，和药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述HTR2b受体激活剂为BW723C86，其用于治疗和/或预防脑缺血再灌注损伤的有效量为1-10mg/kg(体重)，较佳地1-5mg/kg(体重)，最佳地2-4mg/kg(体重)。
  8. 如权利要求6所述的药物组合物，其特征在于，所述治疗和/或预防脑缺血再灌注损伤的其他药物包括：抗ICAM-1抗体、E-选择素、米诺环素、芬戈莫德或其组合。
  9. 一种药盒，其特征在于，所述药盒包含：
    (I)位于第一容器内的第一制剂，所述第一制剂包含(a)HTR2b受体激活剂作为活性成分，和(b)药学上可接受的载体；
    (II)位于第二容器内的第二制剂，所述第二制剂包含治疗和/或预防脑缺血再灌注损伤的其他药物作为活性成分；和
    (III)说明书，所述说明书注明所述药盒用于治疗和/或预防脑缺血再灌注损伤。
  10. 一种体外增强单核巨噬细胞吞噬能力的方法，其特征在于，所述方法包括：向单核巨噬细胞施用HTR2b受体激活剂。
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