CN117085130A - Application of HTR2b activator in improving cerebral ischemia reperfusion injury - Google Patents
Application of HTR2b activator in improving cerebral ischemia reperfusion injury Download PDFInfo
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- CN117085130A CN117085130A CN202210514826.4A CN202210514826A CN117085130A CN 117085130 A CN117085130 A CN 117085130A CN 202210514826 A CN202210514826 A CN 202210514826A CN 117085130 A CN117085130 A CN 117085130A
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- reperfusion injury
- ischemia reperfusion
- cerebral ischemia
- htr2b
- activator
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Classifications
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- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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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
- A61K31/33—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
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- A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/65—Tetracyclines
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- 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
Abstract
The application provides application of an HTR2b activator in preparing a medicament for improving cerebral ischemia reperfusion injury and a method for treating and/or preventing cerebral ischemia reperfusion injury. According to the application, by constructing a cerebral ischemia reperfusion injury mouse model, the up-regulation of the specificity of the peripheral infiltration into the brain of mononuclear/macrophage HTR2b receptor after cerebral ischemia reperfusion injury is determined, and further, the effect that the cerebral infarction area and BBB injury of a mouse can be effectively improved by applying an HTR2b receptor specific agonist (such as BW723C 86) to the cerebral ischemia reperfusion injury mouse model is verified, and a new idea is provided for the immunotherapy of cerebral ischemia reperfusion injury.
Description
Technical Field
The application relates to the field of biological medicine, in particular to application of an HTR2b activator in improving cerebral ischemia reperfusion injury.
Background
Recent global disease burden studies indicate that Ischemic Stroke (Ischemic Stroke) has jumped the first three of the world population disease burden, and Stroke and Ischemic heart disease are the leading causes of death and disability nationwide in China in 2017. Acute ischemic cerebral apoplexy is a common cerebral apoplexy type, accounting for 60% -80% of all cerebral apoplexy. However, according to the new release guidelines for stroke treatment of AHA in 2018, there are no recombinant human tissue plasminogen activator (rt-PA) thrombolysis and intravascular mechanical thrombolytic therapy. With the development of the vascular opening treatment, the damage of cerebral ischemia reperfusion is more and more not ignored. However, no FDA approved pharmaceutical or non-pharmaceutical neuroprotective therapies are currently available to treat reperfusion injury associated with blood flow remodeling or nerve injury outside of the thrombolytic window of time.
The neuroinflammatory reaction after cerebral ischemia reperfusion injury is a great focus in research in recent years, and particularly, the neuroinflammatory reaction research caused by peripheral immune cell infiltration after reperfusion is a great breakthrough in recent years. A large number of immune cells, including blood-derived mononuclear/macrophages, neutrophils and lymphocytes, infiltrate and activate the ischemic areas of the brain following reperfusion, playing a critical role in the development and progression of neuroinflammation. Among these cells, the multiple roles played by macrophages have been of great interest in recent years. Ralf Stumm et al observed that in the acute phase after stroke using Cxcr4-GFP mice, massive mononuclear/macrophages recruited from the bone marrow to migrate through the blood to the brain began to attack adjacent and dead brain tissue; in the subacute stage after cerebral ischemia reperfusion injury, mononuclear/macrophages infiltrated into the brain effectively clear apoptotic neurons in the infarct zone. Therefore, mononuclear/macrophages undergo more complex changes after cerebral ischemia reperfusion injury and play a very important role in prognosis of cerebral ischemia reperfusion injury. However, at present, how peripheral infiltration into brain/mononuclear macrophages affects cerebral ischemic injury remains an unknown problem. However, due to the limitations of the prior art, there is a limited means for differentiating the central intrinsic microglial cells from the peripheral infiltrated mononuclear macrophages after the cerebral ischemia reperfusion injury, so that little is known about how the mononuclear macrophages infiltrated into the brain after the cerebral ischemia reperfusion injury affect the complexity of the brain injury outcome.
Disclosure of Invention
The application aims to provide application of HTR2B activator in preparing medicament for improving cerebral ischemia reperfusion injury and a method for treating and/or preventing cerebral ischemia reperfusion injury.
In a first aspect of the application there is provided the use of an HTR2b receptor or an activator thereof for the preparation of a formulation or composition for:
(i) Up-regulating phagocytic function of mononuclear macrophages; and/or
(ii) Preventing and/or alleviating cerebral ischemia reperfusion injury.
In another preferred embodiment, the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
In another preferred embodiment, the activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof.
In another preferred embodiment, the activator is BW723C86.
In another preferred embodiment, the monocyte macrophage specifically expresses the HTR2b receptor in a high degree.
In another preferred embodiment, the mononuclear macrophage is a mononuclear macrophage that infiltrates into the brain from the periphery.
In another preferred embodiment, the "alleviating cerebral ischemia reperfusion injury" comprises: reduce cerebral infarct size, and/or reduce blood-brain barrier (BBB) damage.
In a second aspect of the present application, there is provided a pharmaceutical composition comprising:
(a) An HTR2b receptor activator, and a pharmaceutically acceptable carrier; and
(b) Other drugs for treating and/or preventing cerebral ischemia reperfusion injury, and pharmaceutically acceptable carriers.
In another preferred embodiment, the pharmaceutical composition is for the treatment and/or prevention of cerebral ischemia reperfusion injury.
In another preferred embodiment, the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is BW723C86 in an effective amount of 1-10mg/kg (body weight), preferably 1-5mg/kg (body weight), and most preferably 2-4mg/kg (body weight) for the treatment and/or prevention of cerebral ischemia reperfusion injury.
In another preferred embodiment, the other drugs for treating and/or preventing cerebral ischemia reperfusion injury include: an anti-ICAM-1 antibody, E-selectin, minocycline, fingolimod, or a combination thereof.
In another preferred embodiment, the pharmaceutical composition is prepared as a liquid formulation or a lyophilized formulation.
In another preferred embodiment, the pharmaceutical composition is prepared as an injection.
In another preferred embodiment, the route of administration of the pharmaceutical formulation comprises: intravenous injection, intravenous infusion, intracranial injection.
In a third aspect of the application, there is provided a kit comprising:
(I) A first formulation within a first container, the first formulation comprising (a) an HTR2b receptor activator as an active ingredient, and (b) a pharmaceutically acceptable carrier;
(II) a second formulation in a second container, the second formulation comprising as an active ingredient a further medicament for the treatment and/or prevention of cerebral ischemia reperfusion injury; and
(III) instructions for use of the kit for treating and/or preventing cerebral ischemia reperfusion injury.
In another preferred embodiment, the first formulation and the second formulation are independent of each other.
In another preferred embodiment, the first formulation is a lyophilized formulation or a liquid formulation, preferably a liquid formulation.
In another preferred embodiment, the first formulation is an injection.
In another preferred embodiment, the first formulation is administered before or after, or simultaneously with, the second formulation.
In another preferred embodiment, the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof, preferably BW723C86.
In another preferred embodiment, the other drugs for treating and/or preventing cerebral ischemia reperfusion injury include: an anti-ICAM-1 antibody, E-selectin, minocycline, fingolimod, or a combination thereof.
In a fourth aspect of the application, there is provided a method of enhancing phagocytic capacity of a mononuclear macrophage in vitro, the method comprising: HTR2b receptor activators are administered to monocytes macrophages.
In another preferred embodiment, the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is BW723C86.
In another preferred embodiment, the mononuclear macrophage is a primary bone marrow-derived macrophage.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In a fifth aspect of the application, there is provided a method of treating and/or preventing cerebral ischemia reperfusion injury, the method comprising the steps of: administering to a subject in need thereof an HTR2b receptor activator, or a pharmaceutical composition according to the second aspect of the application.
In another preferred embodiment, the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof.
In another preferred embodiment, the HTR2b receptor activator is BW723C86.
In another preferred embodiment, the subject in need thereof comprises a human or non-human mammal.
In another preferred embodiment, the cerebral ischemia reperfusion injury is cerebral ischemia reperfusion injury caused by ischemic stroke.
It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows that the peripheral infiltration into the brain of mononuclear macrophage neurotransmitter pathways is specifically enhanced following MCAO. (A) the t-SNE plot was used to identify different models and tissue-derived mononuclear macrophages, with each dot representing a cell. (B) the t-SNE plot was used to identify 13 different clusters into which monocytes were divided, where clusters 3,4 were monocytes that entered the brain from the periphery. (C) The heat map shows the KEGG pathway analysis results for 13 clusters of monocytes macrophages, with the blue boxes indicating a significant enrichment of the 5-HT and GABA receptor pathways of macrophages (clusters 3, 4) entering the brain.
FIG. 2 shows peripheral infiltration into the brain of mononuclear macrophage HTR2B receptor-specific environmental dependence up-regulation. (A) The expression of various neurotransmitter receptor subtypes in different clusters (0-12) of mononuclear macrophages. (B) Immunofluorescence showed HTR2B expression around cerebral infarction in MCAO and Sham mice, scale bar = 50 μm.
Fig. 3 shows that HTR2B receptor specific agonists can modulate phagocytic function of monocytes effective in improving cerebral infarct size and BBB injury in 3d mice following MCAO. (A) Representative MAP2 and IgG staining for mouse cerebral infarction 3 days after cerebral stroke, (B-C) quantification of infarct volume and IgG+ volume, p.ltoreq.0.05, p.ltoreq.0.01.
FIG. 4 shows DEG of primary bone marrow-derived macrophages after (A) administration with HTR2B receptor-specific activator (BW 723C 86) and PBS. (B-C) KEGG pathway and GESA analysis of DEG in A. (D) Immunofluorescence results schematic of in vitro phagocytosis results and determination of phagocytosis index were performed after co-culture with primary bone marrow-derived macrophages and HTR2B receptor specific agonists (BW 723C 86) and inhibitors (SB 204741). Scale = 20 μm, N = 6-9 per group, +.p.ltoreq.0.05, +.p.ltoreq.0.01.
Detailed Description
Through extensive and intensive studies, the inventors of the present application have unexpectedly found that neurotransmitter receptor HTR2b plays an important role in regulating phagocytic function of macrophages and affecting ischemia reperfusion brain injury, and have proposed a novel method for treating brain ischemia reperfusion injury. The inventor confirms that the specificity of peripheral infiltration into brain of mononuclear/macrophage HTR2b receptor is up-regulated through single cell RNA sequencing and immunofluorescence experiments after cerebral ischemia reperfusion injury, and further verifies that administration of HTR2b receptor specific agonist (such as BW723C 86) to a cerebral ischemia reperfusion injury mouse model can effectively improve cerebral infarction area and BBB injury of the mouse. In addition, in vitro experiments also demonstrate that HTR2b receptor specific agonists can significantly enhance phagocytic function of mononuclear/macrophages.
On this basis, the present application has been completed.
Terminology
HTR2b receptors
The 5-HT receptor, also known as serotonin receptor, is a group of G protein-coupled receptors and ligand ion channels found in the nervous system and can be divided into seven subfamilies, 5-HT 1-7. Wherein the family of 5-HT2 receptors includes three subtypes, HTR2a, HTR2b, and HTR2c, wherein HTR2b is a specific receptor for 5-hydroxytryptamine (5-HT) that modulates emotion, behavior, and cognition. The literature suggests that age-dependent upregulation of HTR2b, but not HTR2a, occurs following cerebral ischemia reperfusion injury. Previous studies have found that HTR2b receptor activation may be involved in human macrophage polarization, helping to maintain an anti-inflammatory state. Hajer El eussini et al also found that the presence of HTR2b receptors limited degeneration of spinal cord mononuclear phagocytes and slowed progression of ALS disease.
HTR2b receptor specific activators
The present application provides for the use of HTR2b receptor activators, including but not limited to small molecule compounds, nucleic acids (e.g., DNA, RNA), proteins (e.g., enzymes, antibodies, etc.).
The 5-HT2B receptor is a G protein coupled receptor for the endogenous neurotransmitter serotonin (5-HT). BW723C86 (hydrochloride), a tryptamine analogue, molecular formula C 16 H 19 ClN 2 OS, molecular weight 322.85, is a highly selective 5-HT2B receptor specific agonist and binds poorly to 5-HT2A, 5-HT2C, 5-HT1A and 5-HT1B receptors. BW723C86 is not an amphetamine-type chemical structure, whereas amphetamine molecules, such as fenfluramine, are one of the anorectic agents, and dietary restrictions are associated with prognosis of stroke.
Pharmaceutical compositions of the application
The application provides a pharmaceutical composition which can be used for: (i) up-regulating phagocytic function of mononuclear macrophages; and/or (ii) preventing and/or alleviating cerebral ischemia reperfusion injury, wherein the "alleviating cerebral ischemia reperfusion injury" comprises reducing cerebral infarction area, and/or alleviating blood-brain barrier (BBB) injury.
The pharmaceutical composition of the present application comprises: (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.
In general, HTR2b receptor activators of the present application can be formulated in a nontoxic, inert, and pharmaceutically acceptable carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.
As used herein, "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
The term "pharmaceutically acceptable carrier" refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the composition may contain a liquid, such as water, saline, buffer. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell transfection reagent.
As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal and/or cell.
As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, polysorbate, ethanol, and combinations thereof. In general, the pharmaceutical preparation is to be compatible with the mode of administration, and the pharmaceutical preparation of the present application may be formulated into injection forms, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other auxiliary agents. The pharmaceutical compositions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The medicine composition of the application can also be prepared into a slow release preparation.
In the case of pharmaceutical formulations, a safe and effective amount of the active ingredient combination (including HTR2b receptor activators) is administered to a mammal.
It will be appreciated that the effective amount of the active ingredients of the present application may vary depending on the mode of administration, the severity of the tumor, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the tumor, the weight of the patient, the immune status of the patient, the route of administration, and the like. Specifically, in a preferred embodiment of the application, the "effective amount" of the HTR2b receptor activator BW723C86 is a plasma concentration of 200ng/g (3 mg/kg (body weight) for a single injection may be maintained for 8 hours).
The mode of administration of the pharmaceutical composition of the present application is not particularly limited, and representative examples include (but are not limited to): intravenous injection, intravenous infusion, intracranial injection, subcutaneous injection, intramuscular injection, and the like.
The medicine box of the application
The present application provides a kit comprising:
(I) A first formulation within a first container, the first formulation comprising (a) an HTR2b receptor activator as an active ingredient, and (b) a pharmaceutically acceptable carrier;
(II) a second formulation in a second container, the second formulation comprising as an active ingredient a further medicament for the treatment and/or prevention of cerebral ischemia reperfusion injury; and
(III) instructions for use of the kit for treating and/or preventing cerebral ischemia reperfusion injury.
The first formulation includes (but is not limited to): freeze-dried preparation, liquid preparation (such as injection).
The second formulation includes (but is not limited to): solid (e.g., tablet, capsule, powder), liquid.
Typically, the kit contains one or more (e.g., at least two) unit dosage forms containing an HTR2b receptor activator and one or more (e.g., at least two) unit dosage forms containing other drugs for treating and/or preventing cerebral ischemia reperfusion injury; preferably 4-10 each.
As used herein, the term "unit dosage form" refers to a dosage form that is required for a single administration of a composition for ease of administration, including, but not limited to, various solid agents (e.g., tablets), liquid agents, capsules, sustained release agents.
The description provided by the application can be as follows: the use method of the kit is to use a unit dosage form containing the HTR2b receptor activator and a unit dosage form containing other medicines for treating and/or preventing cerebral ischemia reperfusion injury simultaneously. The kit is particularly suitable for cerebral ischemia reperfusion injury caused by ischemic stroke.
The medicine box provided by the application is prepared by the following steps: the formulation containing the HTR2b receptor activator and the formulation of other drugs for treating and/or preventing cerebral ischemia reperfusion injury are placed together with instructions to form a kit.
The preparation containing the HTR2b receptor activator preferably contains a unit dosage form of the HTR2b receptor activator, and the preparation containing the other medicine for treating and/or preventing cerebral ischemia reperfusion injury preferably contains a unit dosage form of the other medicine for treating and/or preventing cerebral ischemia reperfusion injury.
The step preferably involves placing together at least one unit dosage form containing an HTR2b receptor activator and at least one unit dosage form containing another drug for treating and/or preventing cerebral ischemia reperfusion injury, and instructions to form a kit.
The method of the application
The application also provides a method for enhancing phagocytic capacity of mononuclear macrophages in vitro, which comprises the following steps: HTR2b receptor activators are administered to monocytes macrophages. In a preferred embodiment of the application, phagocytic capacity of the monocular macrophages BMDM is enhanced by administering HTR2b receptor activator BW723C86 to in vitro cultured primary monocular macrophages BMDM (Bone Marrow-Derived Macrophage).
The present application also provides a method of treating and/or preventing cerebral ischemia reperfusion injury, the method comprising the steps of: administering to a subject in need thereof an HTR2b receptor activator, or a pharmaceutical composition according to the second aspect of the application. In a preferred embodiment of the present application, the phagocytic capacity of mononuclear macrophages infiltrating the brain from the periphery is enhanced by administering the HTR2b receptor activator BW723C86 to a test animal (ischemic stroke model mouse), thereby improving the cerebral infarct size and BBB injury of the mouse.
The beneficial effects of the application include:
1) Revealing a novel mechanism of interaction between the nervous system and peripheral infiltrated mononuclear macrophages after cerebral ischemia reperfusion injury, and revealing a novel mechanism of HTR2b receptor regulating phagocytic function of the mononuclear macrophages;
2) Provides a new strategy for specifically activating HTR2b receptor in early stage after cerebral ischemia reperfusion injury (BW 723C86 is given), provides a new thought for the immunotherapy of cerebral ischemia reperfusion injury, improves the life quality of patients, and reduces the burden of families and society.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally in accordance with conventional regulations, such as those described in J.Sambrook et al, molecular cloning, an experimental guideline (fourth edition) (science Press, inc., 2017), or in accordance with the specifications provided by the manufacturer of the product. Materials, reagents, instruments and the like used in the examples are commercially available unless otherwise specified.
Experimental method
1. Establishment of ischemic cerebral apoplexy model
MCAO molding was performed on 8-to 10-week-old male wild-type mice in a C57BL/6 background. Briefly, first with 30% O 2 /70％N 2 The mice were anesthetized by mixing 2% isoflurane with the mixed air. After complete anesthesia, a skin incision was made in the mouse neck, exposing the left common carotid artery and its branches, and temporarily ligating the common carotid artery, followed by the truncation of the external carotid artery. And inserting a wire plug from the external carotid artery into the internal carotid artery through the residual of the external carotid artery, and inducing MCAO by endoluminal 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 operation, the temperature of the mice is ensured by using the heating padIs maintained at 37+/-0.5 ℃.
2. Single cell RNA-seq experiments
Cell lysis buffer was added to allow hybridization of polyadenylated RNA molecules to the microbeads. The beads were collected into a single tube for reverse transcription. After cDNA synthesis, each BD Rhapsody system was used to obtain single cell transcriptome information. Single cell capture was achieved using a limited dilution method by randomly distributing single cell suspensions in >200,000 microwells. Beads with oligonucleotide codes are added to saturation to pair the beads with cells in the microwells. The cDNA molecules are labeled at the 5 'end (i.e., the 3' end of the mRNA transcript) with a Unique Molecular Identifier (UMI) and a cell label indicating the cell from which they were derived. Complete transcriptome libraries were prepared using the BD Rhapsody single cell whole transcriptome amplification workflow. Briefly, second strand cDNA was synthesized and then ligated to WTA adaptors for universal amplification. 18 PCR cycles were used to amplify the adaptor-ligated cDNA product. A sequencing library for the whole transcriptome amplification product was prepared using random primer PCR to enrich for 3' ends of transcripts linked to cell markers and UMI. Sequencing libraries were quantified using a high sensitivity DNA chip (agilent) on Bioanalyzer2200 and a Qubit high sensitivity DNA analysis (Thermo Fisher Scientific). The library of each sample was sequenced by HiSeq Xten (I-coil, san Diego, calif.) with paired end sequencing of 150 bp.
3. Measurement of infarct volume
Cerebral infarct size was measured using microtubule-associated protein 2 (MAP-2) staining, animals were sacrificed and perfused transcardiac with PBS containing 0.9% physiological saline and 4% paraformaldehyde. 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 (1.52 a) analysis by a viewer with an unknown group of experimental groups.
4. Isolation and culture of Primary macrophage BMDM (Bone Marrow-Derived Macrophage)
1) Leg bone is got to mouse
The mice were sacrificed after anesthesia, the mice were placed in beakers containing a sufficient amount of 75% ethanol for 5min of sterilization, and the soaked animals were blotted with paper to remove excess ethanol. A small opening is cut on the back of the mouse by using scissors, and the skin is directly torn to the joints of the lower leg of the mouse by hands, so that the joints and the skin of the foot of the mouse are removed (the process slightly shows rough blood fishy smell but has high efficiency). The hind limbs are detached (the legs are not broken off by the scissors) along the root of the thigh of the mouse, muscle tissues are removed, the hind limbs are placed in a culture dish containing 75% ethanol for soaking for 5min, and a new culture dish containing 75% ethanol is replaced and is moved into an ultra clean bench.
2) BMDM cell extraction and induction
The ethanol soaked leg bones are moved into cold PBS for soaking, the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The washed femur and tibia are separated, two ends of the femur and tibia are cut off respectively by scissors, the bone marrow is blown out of the femur and tibia by sucking cold induction culture medium by a 1mL syringe, and the flushing is repeated for 3 times until no obvious red color is seen in the femur. Repeatedly blowing the culture medium containing bone marrow cells with a 5mL pipetting gun to disperse cell clusters, sieving the cells with a 70 μm cell filter, transferring into a 15mL centrifuge tube, centrifuging at 1500rpm/min for 5min, discarding the supernatant, adding erythrocyte lysate, re-suspending for 5min, centrifuging at 1500rpm/min for 5min, discarding the supernatant, re-suspending with cold prepared bone marrow macrophage induced culture medium, and plating. The culture medium is not replaced during the cell culture period, half of the bone marrow macrophage induction culture medium is replaced after the third day of culture, all the culture medium is replaced in the fifth day, and the seventh day can be used for the subsequent experiments.
5. Phagocytosis experiment
BMDM cells were grown at 2X 10 4 The density of individual cells/wells was spread on a 1.5-mm2 coverslip for 24 hours. Then, the fluorescent-labeled latex beads were added at a concentration of 5. Mu.l/ml for 2 hours at 37 ℃. Cells were washed 3 times with PBS to remove non-phagocytized beads and fixed with 4% paraformaldehyde. Next, phagocytosis of the beads by microglial cells was observed under a fluorescent inverted microscope. Cells with phagocytic activity were observed using fluorescence microscopy (magnification x 40 and x 100). All experiments were repeated 3 times. Negative control microglial cells were pre-conditioned with cytochalasin D (10. Mu.M)Treating for 30min.
6. Statistical method
All statistical data were performed using GraphPad Prism v.6 or the implemented statistical tests of the corresponding R-packets. The distribution was tested for normality and equality of variance before any parametric analysis was applied. The two-tailed student t-test was used for pairwise comparison between the two groups. The remaining data were analyzed using one-way or two-way ANOVA as appropriate. Multiple comparison procedures were performed using Bonferroni's post hoc test to determine specific group differences. Results are expressed as mean ± standard deviation. Correlation analysis was performed using Pearson correlation analysis. P.ltoreq.0.05 is considered statistically significant.
Example 1 single cell RNA sequencing shows that peripheral infiltration into the brain has been specifically enhanced by the neurotransmitter pathway of mononuclear macrophages
To explore the complex changes of mononuclear/macrophages infiltrating into the brain from the periphery after cerebral ischemia reperfusion injury, single cell sequencing was performed on cerebral infarct tissue of mice 3 days after establishment of middle cerebral artery occlusion Model (MCAO), and deep analysis was performed using macrophage marker-Ms 4a7, single-core macrophages at specific markers and single-core/macrophages in bone marrow using the method of RacID 3.
The results show that a significantly specific enrichment of neurotransmitter receptor pathways, in particular the 5-HT and GABA receptor pathways, occurs in the peripheral infiltration into the brain of mononuclear/macrophages following cerebral ischemia reperfusion injury relative to the mononuclear/macrophages in the bone marrow (fig. 1).
EXAMPLE 2 peripheral infiltration into the brain monocyte macrophage HTR2b receptor specific Environment dependent upregulation
Based on the discovery of specific enrichment of neurotransmitter receptor pathways into the brain by mononuclear macrophages, various neurotransmitter receptor subtypes were analyzed, and the discovery of high expression of HTR2b receptor specificity by macrophages (clusters 3, 4) peripherally entering the brain (fig. 2A) suggests that high expression of HTR2b receptor specificity by peripheral mononuclear/macrophages may play a very important role in cerebral ischemia reperfusion injury.
To verify the changes in HTR2b receptor expression observed in single cell sequencing at the protein level, weThe method of co-labeling by over-immunofluorescence verifies F4/80 in the brain of 3d mice after MCAO + Is significantly increased in macrophages (fig. 2B).
The above experimental results suggest that neurotransmitter receptor HTR2b expression by peripheral mononuclear macrophages infiltrated into the brain is significantly up-regulated following cerebral ischemia reperfusion injury.
Example 3 htr2b receptor-specific agonists are effective in improving cerebral infarct size and BBB injury in 3d mice following MCAO
To investigate the effect of HTR2b receptors of mononuclear macrophages infiltrating the brain on brain ischemia reperfusion injury, it was found that HTR2b receptor specific agonists (BW 723C 86) could effectively improve cerebral infarct size and BBB injury in mice 3 days after MCAO by 2 hours after MCAO (fig. 3), indicating that upregulation of HTR2b receptors of mononuclear macrophages may be a spontaneous protective mechanism following brain ischemia reperfusion injury, which the body produces, and could alleviate brain ischemia reperfusion injury.
Example 4 htr2b receptor-specific agonists can modulate phagocytosis of monocytes
To elucidate the mechanism of HTR2b receptor protection against ischemic cerebral ischemia reperfusion injury, changes in the transcriptome of primary bone marrow-derived macrophages following administration of HTR2b receptor-specific activator (BW 723C 86) were examined, and both KEGG and GESA assays found a significant enrichment of phagocytic pathways following administration of BW723C86 (fig. 4A-C).
To determine whether HTR2b receptor protection mechanisms against ischemic brain ischemia reperfusion injury works by altering phagocytic function of monocytes macrophages, agonists were administered to primary bone marrow-derived macrophages following co-culture with dose-dependent HTR2b receptor specific agonists (BW 723C 86) and inhibitors (SB 204741) to significantly enhance phagocytic function of primary macrophages BMDM (fig. 4D).
The above results indicate that HTR2b receptors regulate the outcome of cerebral ischemia reperfusion injury by affecting phagocytic function of macrophages.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. Use of an HTR2b receptor, or an activator thereof, for the preparation of a formulation or composition for:
(i) Up-regulating phagocytic function of mononuclear macrophages; and/or
(ii) Preventing and/or alleviating cerebral ischemia reperfusion injury.
2. The use of claim 1, wherein the activator comprises a small molecule compound, a nucleic acid, a protein, or a combination thereof.
3. The use according to claim 1, wherein the activator is selected from the group consisting of: BW723C86, 5-HT, or a combination thereof.
4. The use of claim 1, wherein the mononuclear macrophages are peripheral infiltration into the brain.
5. The use of claim 1, wherein said reducing brain ischemia reperfusion injury comprises: reduce cerebral infarct size, and/or reduce blood-brain barrier (BBB) damage.
6. A pharmaceutical composition, comprising:
(a) An HTR2b receptor activator, and a pharmaceutically acceptable carrier; and
(b) Other drugs for treating and/or preventing cerebral ischemia reperfusion injury, and pharmaceutically acceptable carriers.
7. Pharmaceutical composition according to claim 6, wherein the HTR2b receptor activator is BW723C86 in an effective amount for the treatment and/or prevention of cerebral ischemia reperfusion injury of 1-10mg/kg (body weight), preferably 1-5mg/kg (body weight), most preferably 2-4mg/kg (body weight).
8. The pharmaceutical composition of claim 6, wherein the additional agent for treating and/or preventing cerebral ischemia reperfusion injury comprises: an anti-ICAM-1 antibody, E-selectin, minocycline, fingolimod, or a combination thereof.
9. A kit, comprising:
(I) A first formulation within a first container, the first formulation comprising (a) an HTR2b receptor activator as an active ingredient, and (b) a pharmaceutically acceptable carrier;
(II) a second formulation in a second container, the second formulation comprising as an active ingredient a further medicament for the treatment and/or prevention of cerebral ischemia reperfusion injury; and
(III) instructions for use of the kit for treating and/or preventing cerebral ischemia reperfusion injury.
10. A method of enhancing phagocytic capacity of a mononuclear macrophage in vitro, comprising: HTR2b receptor activators are administered to monocytes macrophages.
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| CN105561313B (en) * | 2016-01-25 | 2018-12-25 | 北京大学 | It reduces 5-HT2BR content and its active substance is inhibited to treat and prevent the application in atherosclerosis product in preparation |
| CN108159394B (en) * | 2018-02-07 | 2020-04-03 | 天津医科大学总医院 | Application of PR-957 in preparing medicine for treating cerebral ischemia reperfusion injury |
| CN112370436B (en) * | 2020-11-19 | 2022-04-29 | 东南大学 | Nano-medicine for preventing or treating cerebral ischemia-reperfusion injury and preparation method and application thereof |
-
2022
- 2022-05-11 CN CN202210514826.4A patent/CN117085130A/en active Pending
-
2023
- 2023-05-11 WO PCT/CN2023/093663 patent/WO2023217249A1/en unknown
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