WO2023023994A1 - 一种提高抑炎型巨噬细胞趋化的药物及其应用 - Google Patents

一种提高抑炎型巨噬细胞趋化的药物及其应用 Download PDF

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WO2023023994A1
WO2023023994A1 PCT/CN2021/114616 CN2021114616W WO2023023994A1 WO 2023023994 A1 WO2023023994 A1 WO 2023023994A1 CN 2021114616 W CN2021114616 W CN 2021114616W WO 2023023994 A1 WO2023023994 A1 WO 2023023994A1
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macrophages
chemotaxis
inflammatory
glucosamine
improving
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陈维倩
周文静
丁靓
沈振亚
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苏州大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

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  • the invention belongs to the pharmaceutical technology, and in particular relates to the application of a compound in the preparation of drugs for improving the chemotaxis of anti-inflammatory macrophages.
  • Macrophages Stimulation of macrophages (macrophages) by IFN- ⁇ or IL-4 exhibits distinct functions. Macrophages stimulated with IFN- ⁇ differentiate into M1 macrophages that promote inflammation, and IL-4 stimulated M2 macrophages that inhibit inflammation. This functional pluripotency is of great significance to various diseases. For example, if macrophages in tumor tissue differentiate into M1 macrophages, they will promote the body’s immune cells to clear cancer cells. On the contrary, if If macrophages differentiate into M2 macrophages, they will suppress the body's immunity.
  • CX3C chemokine ligand 1 [CX3CL1] is CX3CR1, which plays an important role in the migration, proliferation and angiogenesis of vascular endothelial cells.
  • CX3CL1 appears as two distinct isoforms: membrane-bound and soluble (sFKN). Thus, it combines the properties of chemoattractants and adhesion molecules.
  • sFKN membrane-bound and soluble
  • the existence of two forms of CX3CL1 in vivo indicates its special role in the large family of chemokines, which is unmatched by any other chemokines so far. Unlike other chemokines that are abundantly expressed in peripheral blood cells, CX3CL1 is extremely Little or no expression in peripheral blood cells.
  • the invention discloses a new application of using GlcN to improve the chemotactic ability of anti-inflammatory macrophages.
  • Macrophages regulate the proliferation and differentiation of vascular endothelial cells in the microenvironment through chemotaxis and secrete cytokines, affecting the occurrence of new blood vessels.
  • Chemokines play an indirect role in regulating angiogenesis by binding to corresponding receptors on the surface of macrophages.
  • the present invention adopts the following technical solutions: A drug for improving chemotaxis of anti-inflammatory macrophages, the active ingredient of which is glucosamine.
  • glucosamine in the preparation of drugs or health products for improving anti-inflammatory macrophage chemotaxis.
  • the invention discloses a method for improving the chemotaxis of anti-inflammatory macrophages to CX3CL1, which uses glucosamine to stimulate the macrophages to improve the chemotaxis of anti-inflammatory macrophages to CX3CL1. Further, glucosamine and cytokines were used to stimulate macrophages to improve the chemotaxis of anti-inflammatory macrophages to CX3CL1.
  • the invention discloses the application of glucosamine and cytokines in the preparation of drugs for improving chemotaxis of anti-inflammatory macrophages to CX3CL1.
  • the cytokine is IL-4 or IL-13.
  • Glucosamine is an amino-containing sugar in the human body, widely present in articular cartilage and connective tissue, and is the basic component of cartilage matrix and joint fluid.
  • glucosamine As an over-the-counter drug for the treatment of osteoarthritis, glucosamine has been used in Europe for more than 20 years, and no obvious side effects have been found in long-term use.
  • Glucosamine has been approved by many countries as a dietary supplement for alleviating arthritis, but so far there has been no research report on glucosamine improving anti-inflammatory macrophage chemotaxis.
  • the present invention treats macrophages with IL-4 or IL-13 to induce polarization, and simultaneously stimulates them with PBS or GlcN for 24 hours.
  • the same number of cells in each group is inoculated into the upper chamber of the cell chemotaxis (transwell), and the lower chamber
  • the chamber is the basal medium supplemented with CX3CL1.
  • wipe the non-chemotactic cells on the upper part of the chamber fix with 4% PFA for 15 minutes, wash twice with PBS, add DAPI-containing anti-fluorescence attenuation agent at the bottom of the chamber, and invert the fluorescence microscope. Take pictures, and imageJ counts the number of cells in each field of view under a 10x microscope, and observes the fluorescence of each group under a fluorescence microscope.
  • Figure 1 shows that glucosamine treatment significantly increased the chemotaxis of anti-inflammatory macrophages to CX3CL1.
  • Figure 2 shows that glucosamine treatment does not affect the pro-inflammatory polarization of macrophages.
  • Figure 3 shows that glucosamine treatment does not affect anti-inflammatory polarization of macrophages.
  • the main materials and sources used are as follows: laser confocal microscope (LSM880, Zeiss); flow cytometer (Millipore Guava easyCyte); real-time fluorescent quantitative PCR instrument (ABI Stepone Plus); ultra-micro spectrophotometer (Nanodrop2000); Fluorescence microscope (Olympus, IX51); Glucosamine (Sigma, U.S.); LPS (Sigma, U.S.); IFN- ⁇ (PeproTech, U.S.); IL-4 (PeproTech, U.S.); IL-13 (PeproTech, U.S.); CX3CL1 (PeproTech, USA); iNOS-PE antibody (Biolegend, USA); CD80-PE antibody (Biolegend, USA); CD86-PE antibody (Biolegend, USA); CD117-PE antibody (Biolegend, USA); transwell chambers ( 8.0- ⁇ m pore size, Corning).
  • the specific experimental method and testing method of the present invention are conventional techniques, and the
  • Example 1 Glucosamine treatment significantly increased the chemotaxis of anti-inflammatory macrophages to CX3CL1: macrophages (iBMDM) were treated with IL-4 (40 ng/mL) and treated with PBS buffer or GlcN (3 mM) respectively Stimulate for 24 hours, specifically: experimental group: add IL-4 and GlcN to RPMI 1640 basal medium, then inoculate macrophages, and conventionally culture (37°C, 5% CO 2 ) for 24 hours; control group: add IL-4 , PBS was added to RPMI 1640 basal medium, and macrophages were inoculated, and cultured for 24 hours.
  • IL-4 40 ng/mL
  • PBS buffer or GlcN 3 mM
  • Embodiment two Macrophages (iBMDM) were treated with IL-13 (20 ng/mL) and stimulated with PBS buffer or GlcN (3 mM) for 24 hours, specifically: experimental group: IL-13 and GlcN were added to RPMI 1640 basal medium In the medium, macrophages were re-inoculated, and conventionally cultured for 24 hours; in the control group: IL-13 and PBS were added to RPMI 1640 basal medium, and macrophages were re-inoculated, and conventionally cultured for 24 hours.
  • Example 1 a routine cell chemotaxis (transwell method) experiment was performed according to Example 1, and the results are shown in FIG. 1 .
  • Macrophages were treated with LPS/IFN- ⁇ (20 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 hours respectively, according to Carry out F4/80 and iNOS immunofluorescence staining by routine steps, add Alexa Fluor 594-labeled and Alexa Fluor 488-labeled fluorescent secondary antibodies, mount with DAPI-containing anti-fluorescence attenuation mounting medium, observe and take pictures of iNOS signals simultaneously with a confocal microscope (green), F4/80 signal (red) and DAPI signal (blue).
  • iBMDM Macrophages
  • Macrophages were treated with LPS/IFN- ⁇ (20 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 hours, collected in 1.5mL EP tubes, and iNOS, CD80 and CD86 flow antibodies were added Incubate at room temperature for 30 min, detect by flow cytometry, and analyze the experimental results with FlowJo software. Referring to Figure 2B, C, D, the results of flow cytometry showed that the average fluorescence intensity of iNOS+, CD80+, CD86+ in the GlcN group had almost no difference from that in the PBS group. Photos shown are representative pictures of samples from each group.
  • Macrophages were treated with LPS/IFN- ⁇ (20 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 hours respectively.
  • RNA was extracted, reverse transcription and QPCR were performed according to conventional procedures, and 18S was used as an internal reference.
  • FIG. 2E which is a graph showing the expression levels of pro-inflammatory cytokines in macrophages detected by QPCR, it can be seen that there is almost no difference between the GlcN group and the PBS group.
  • Example 4 Glucosamine treatment does not affect the anti-inflammatory polarization of macrophages Macrophages (iBMDM) were treated with IL-4 (40 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 hours respectively, according to conventional procedures Perform F4/80 and CD206 immunofluorescence staining, add Alexa Fluor 594-labeled and Alexa Fluor 488-labeled fluorescent secondary antibodies, mount with DAPI-containing anti-fluorescence attenuation mounting medium, observe and take pictures of CD206 signals simultaneously with a confocal microscope (green ), F4/80 signal (red) and DAPI signal (blue). Referring to FIG.
  • Macrophages were treated with IL-4 (40 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 h, collected in 1.5 mL EP tubes, and incubated with CD206 flow cytometry antibody for 30 min at room temperature, and analyzed by flow cytometry Detection was carried out, and FlowJo software was used to analyze the experimental results.
  • FIG. 3B the results of flow cytometry showed that the average fluorescence intensity of CD206+ in the GlcN group was almost indistinguishable from that in the PBS group. Photos shown are representative pictures of samples from each group.
  • Macrophages were treated with IL-4 (40 ng/mL) and stimulated with PBS or GlcN (3 mM) for 24 h, and RNA was extracted, reverse transcribed and QPCR were performed according to conventional procedures, and 18S was used as an internal reference.
  • FIG. 3C which is a graph showing the expression levels of pro-inflammatory and anti-inflammatory cytokines in macrophages detected by QPCR, it can be seen that there is almost no difference between the GlcN group and the PBS group.

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Abstract

一种提高抑炎型巨噬细胞趋化的药物及其在制备改善抑炎型巨噬细胞趋化药物中的应用，所述药物的活性成分为葡萄糖胺。体外实验表明，葡萄糖胺体外处理不影响巨噬细胞促炎型或抑炎型极化，能提高抑炎型巨噬细胞对CX3CL1的趋化性。

Description

一种提高抑炎型巨噬细胞趋化的药物及其应用 技术领域
本发明属于药物技术，具体涉及一种化合物在制备改善抑炎型巨噬细胞趋化的药物中的应用。
 背景技术
巨噬细胞（macrophages）通过IFN-γ或者IL-4刺激会表现出截然不同的功能。刺激了IFN-γ的巨噬细胞会分化成促进炎症的M1型巨噬细胞，刺激了IL-4会表现出抑制炎症的M2型巨噬细胞。这一功能上的多能性对于各种疾病有重要的意义，比如，在肿瘤组织中的巨噬细胞如果分化为M1型巨噬细胞就会促进机体的免疫细胞对于癌细胞的清除，相反如果巨噬细胞如果分化为M2型巨噬细胞就会抑制机体免疫力。CX3C趋化因子配体1[CX3CL1]的受体是CX3CR1，在血管内皮细胞迁移、增生以及血管发生中均发挥重要作用。在体内，CX3CL1以两种不同的亚型出现：膜结合型和可溶型（sFKN）。因此，它结合了趋化剂和粘附分子的特性。体内CX3CL1两种形式的存在说明它在趋化因子大家族中的特殊作用，这是迄今为止任何其他趋化因子都无法比拟的，与其他趋化因子丰富表达于外周血细胞的情况不同，CX3CL1极少甚至不表达于外周血细胞。
 技术问题
本发明公开了一种利用GlcN改善抑炎型巨噬细胞趋化能力的新用途。巨噬细胞通过趋化并分泌细胞因子来调节微环境中血管内皮细胞的增生和分化，影响新生血管的发生，趋化因子通过结合巨噬细胞表面相应受体发挥间接调节血管生成的作用。
 技术解决方案
本发明采用如下技术方案： 一种提高抑炎型巨噬细胞趋化的药物，所述药物的活性成分为葡萄糖胺。
葡萄糖胺在制备改善抑炎巨噬细胞趋化的药物或保健品中的应用。
葡萄糖胺在制备改善抑炎巨噬细胞向CX3CL1趋化的药物或保健品中的应用。
本发明公开了一种改善抑炎巨噬细胞向CX3CL1趋化的方法，利用葡萄糖胺刺激巨噬细胞，改善抑炎巨噬细胞向CX3CL1的趋化。进一步的，利用葡萄糖胺与细胞因子刺激巨噬细胞，改善抑炎巨噬细胞向CX3CL1的趋化。
本发明公开了葡萄糖胺与细胞因子在制备改善抑炎巨噬细胞向CX3CL1趋化的药物中的应用。
本发明中，细胞因子为IL-4或IL-13。
 有益效果
葡萄糖胺（glucosamine，GlcN）是人体内一种含氨基的糖，广泛存在于关节软骨与结缔组织中，是软骨基质和关节液的基本成分。作为治疗变形性关节炎的非处方类药物，葡萄糖胺已在欧洲应用二十余年，长期使用未发现明显副作用。葡萄糖胺已经被多个国家批准为缓解关节炎的膳食补充剂，但迄今为止尚未见葡萄糖胺改善抑炎巨噬细胞趋化方面的研究报道。本发明用IL-4或IL-13处理巨噬细胞诱导极化，同时分别用PBS或GlcN刺激24h，细胞计数后将每组同样数量的细胞接种到细胞趋化（transwell）上室中，下室为添加CX3CL1的基础培养基，继续常规培养4h后，擦拭小室上部未趋化的细胞，4%PFA固定15min，PBS清洗2次，小室底部滴加含DAPI的抗荧光衰减剂，倒置荧光显微镜拍照，imageJ统计10倍镜下每个视野细胞数量，荧光显微镜下观察各组荧光，发现GlcN防治组趋化巨噬细胞数量显著多于PBS组。证实了葡萄糖胺改善抑炎巨噬细胞向CX3CL1趋化的能力。实验的结果综合证明：GlcN体外处理并不影响巨噬细胞促炎型或抑炎型极化，但显著提高抑炎型巨噬细胞的趋化率。
 附图说明
图1表示葡萄糖胺处理显著增加抑炎型巨噬细胞对CX3CL1的趋化性。
图2表示葡萄糖胺处理不影响巨噬细胞促炎型极化。
图3表示葡萄糖胺处理不影响巨噬细胞抑炎型极化。
 本发明的实施方式
以下所列实施例，仅为帮助本领域技术人员更全面理解本发明，但不以任何方式限制本发明。
所用的主要材料和来源分别如下：激光共聚焦显微镜（LSM880，Zeiss）；流式细胞仪（Millipore Guava easyCyte）；实时荧光定量PCR仪（ABI Stepone Plus）；超微量分光光度计（Nanodrop2000）；倒置荧光显微镜 (Olympus，IX51)；葡萄糖胺（Sigma，美国）；LPS（Sigma，美国）；IFN-γ（PeproTech，美国）；IL-4（PeproTech，美国）；IL-13 (PeproTech，美国) ；CX3CL1（PeproTech，美国）；iNOS-PE抗体（Biolegend，美国）；CD80-PE抗体（Biolegend，美国）；CD86-PE抗体（Biolegend，美国）；CD117-PE抗体（Biolegend，美国）；transwell chambers (8.0-μm 孔径, Corning)。本发明具体实验方法与测试方法为常规技术，实验组与对照组为平行实验。
实施例一 葡萄糖胺处理显著增加抑炎型巨噬细胞对CX3CL1的趋化性：用IL-4（40 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS缓冲液或GlcN（3 mM）刺激24h，具体的：实验组：将IL-4、GlcN加入RPMI 1640基础培养基中，再接种巨噬细胞，常规培养（37℃、5%CO 2）24小时；对照组：将IL-4、PBS加入RPMI 1640基础培养基中，再接种巨噬细胞，常规培养24小时。
然后进行常规细胞趋化（transwell法）实验，具体的：细胞计数后将每组同样数量的细胞分别接种到transwell上室中，下室为添加CX3CL1的RPMI 1640基础培养基，继续常规培养4h后，擦拭小室上部未趋化的细胞，4%PFA固定15min，PBS清洗2次，小室底部滴加含DAPI的抗荧光衰减剂，倒置荧光显微镜拍照，imageJ统计10倍镜下每个视野细胞数量，结果见图1。
实施例二： 用IL-13（20 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS缓冲液或GlcN（3 mM）刺激24h，具体的：实验组：将IL-13、GlcN加入RPMI 1640基础培养基中，再接种巨噬细胞，常规培养24小时；对照组：将IL-13、PBS加入RPMI 1640基础培养基中，再接种巨噬细胞，常规培养24小时。
然后按照实施例一进行常规细胞趋化（transwell法）实验，结果见图1。
参见图1A，荧光显微镜下观察各组荧光，发现GlcN防治组趋化巨噬细胞数量显著多于PBS组，照片显示的是每组样本中的代表性图片，标尺显示100 µm，图1B为统计后的数据。
实施例三 葡萄糖胺处理不影响巨噬细胞促炎型极化： 用LPS/IFN-γ（20 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS或GlcN（3 mM）刺激24h，按常规步骤进行F4/80和iNOS免疫荧光染色，添加Alexa Fluor 594标记和Alexa Fluor 488标记的荧光二抗，用含DAPI的抗荧光衰减封片剂封片，以共聚焦显微镜同时观察并拍照iNOS信号（绿），F4/80信号（红）和DAPI信号（蓝）。参见图2A，激光共聚焦显微镜下观察各组荧光，发现GlcN组的F4/80+iNOS+巨噬细胞数量与PBS组几乎没有差异。照片显示的是每组样本中的代表性图片，标尺显示20 µm。
用LPS/IFN-γ（20 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS或GlcN（3 mM）刺激24h，收集于1.5mL EP管中，分别加入iNOS、CD80及CD86流式抗体室温孵育30min,通过流式细胞仪进行检测，FlowJo软件分析实验结果。参见图2B、C、D，流式细胞术结果显示，GlcN组的iNOS+、CD80+、CD86+的平均荧光强度与PBS组几乎没有差异。照片显示的是每组样本中的代表性图片。
用LPS/IFN-γ（20 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS或GlcN（3 mM）刺激24h，按常规步骤提取RNA、逆转录和QPCR，以18S作为内参。参见图2E，为QPCR检测巨噬细胞促炎型细胞因子表达水平图，可以看出，GlcN组与PBS组几乎没有差异。
实施例四 葡萄糖胺处理不影响巨噬细胞抑炎型极化： 用IL-4（40 ng/mL）处理巨噬细胞（iBMDM）同时分别用PBS或GlcN（3 mM）刺激24h，按常规步骤进行F4/80和CD206免疫荧光染色，添加Alexa Fluor 594标记和Alexa Fluor 488标记的荧光二抗，用含DAPI的抗荧光衰减封片剂封片，以共聚焦显微镜同时观察并拍照CD206信号（绿），F4/80信号（红）和DAPI信号（蓝）。参见图3A，激光共聚焦显微镜下观察各组荧光，发现GlcN组的F4/80+CD206+巨噬细胞数量与PBS组几乎没有差异。照片显示的是每组样本中的代表性图片，标尺显示20 µm。
用IL-4（40 ng/mL）处理巨噬细胞同时分别用PBS或GlcN（3 mM）刺激24h，收集于1.5mL EP管中，分别加入CD206流式抗体室温孵育30min,通过流式细胞仪进行检测，FlowJo软件分析实验结果。参见图3B，流式细胞术结果显示，GlcN组的CD206+的平均荧光强度与PBS组几乎没有差异。照片显示的是每组样本中的代表性图片。
用IL-4（40 ng/mL）处理巨噬细胞同时分别用PBS或GlcN（3 mM）刺激24h，按常规步骤提取RNA、逆转录和QPCR，以18S作为内参。参见图3C，为QPCR检测巨噬细胞促炎抑炎型细胞因子表达水平图，可以看出，GlcN组与PBS组几乎没有差异。
【结论】上述实验的结果综合证明：GlcN体外处理并不影响巨噬细胞促炎型或抑炎型极化，但显著提高抑炎型巨噬细胞的趋化率。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种提高抑炎型巨噬细胞趋化的药物，其特征在于，所述药物的活性成分为葡萄糖胺。
  2. 葡萄糖胺在制备改善抑炎巨噬细胞向CX3CL1趋化的药物中的应用。
  3. 葡萄糖胺在制备改善抑炎巨噬细胞趋化的药物中的应用。
  4. 葡萄糖胺在制备改善抑炎巨噬细胞向CX3CL1趋化的保健品中的应用。
  5. 葡萄糖胺在制备改善抑炎巨噬细胞趋化的保健品中的应用。
  6. 根据权利要求2、3、4或者5所述的应用，其特征在于，所述药物或者保健品为液体制剂。
  7. 一种改善抑炎巨噬细胞向CX3CL1趋化的方法，其特征在于，利用葡萄糖胺刺激巨噬细胞，改善抑炎巨噬细胞向CX3CL1的趋化。
  8. 根据权利要求7所述的方法，其特征在于，利用葡萄糖胺与细胞因子刺激巨噬细胞，改善抑炎巨噬细胞向CX3CL1的趋化。
  9. 根据权利要求8所述的方法，其特征在于，细胞因子为IL-4或IL-13。
  10. 葡萄糖胺与细胞因子在制备改善抑炎巨噬细胞向CX3CL1趋化的药物中的应用。
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CN107771080A (zh) * 2015-04-08 2018-03-06 微脉管健康管理有限公司 协同糖萼治疗组合物和方法
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