WO2022052234A1 - 一种调节巨噬细胞极化状态的方法 - Google Patents

一种调节巨噬细胞极化状态的方法 Download PDF

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WO2022052234A1
WO2022052234A1 PCT/CN2020/124288 CN2020124288W WO2022052234A1 WO 2022052234 A1 WO2022052234 A1 WO 2022052234A1 CN 2020124288 W CN2020124288 W CN 2020124288W WO 2022052234 A1 WO2022052234 A1 WO 2022052234A1
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macrophages
electrical stimulation
stimulation signal
cholinergic
inflammatory
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钱政江
李洪超
杨海洋
李翔
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Shenzhen Institute of Advanced Technology of CAS
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36014—External stimulators, e.g. with patch electrodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/3605—Implantable neurostimulators for stimulating central or peripheral nerve system
    • A61N1/36053—Implantable neurostimulators for stimulating central or peripheral nerve system adapted for vagal stimulation
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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 application belongs to the technical field of biomedicine, and relates to a method for regulating the polarization state of macrophages, in particular to a method for regulating the polarization state of macrophages by activating the cholinergic anti-inflammatory pathway and its effect in delaying the occurrence of atherosclerotic inflammation developing applications.
  • Macrophages are formed by the differentiation of myeloid progenitor cells into monocytes, which then enter the blood and migrate into tissues for differentiation. According to the difference of activation mode and function, macrophages are divided into classic type (M1 type) and alternative type (M2 type).
  • macrophages Under the stimulation of bacterial toxin LPS or Th1 cytokines (TNF ⁇ , IFN ⁇ ), macrophages are activated and polarized into M1 type, express and release pro-inflammatory factors and reactive oxygen species, which can kill pathogens, clear necrotic tissue and promote The role of inflammatory response; correspondingly, under the induction of Th2 cytokines (IL-4, IL-13), macrophages are activated to M2 type and express anti-inflammatory factors (such as TGF ⁇ , IL-10), Has the effect of reducing inflammation, promoting tissue growth and repair.
  • the polarization state of macrophages can also be regulated by changing the expression of Krüppel-like factors (KLF4), STAT6, miR-33 and other genes.
  • the Cholinergic Anti-inflammatory Pathway is a newly discovered neuroimmunomodulatory anti-inflammatory pathway. Under inflammatory conditions, the afferent vagus (afferent vagus) transmits inflammatory signals to the central nervous system, and then the efferent vagus (efferent vagus) releases acetylcholine (ACh) to act on macrophages, thereby reducing the release of cellular inflammatory factors , can effectively inhibit the development of inflammatory response.
  • CAP has obvious inhibitory effects on both systemic and local inflammation, providing an effective way to regulate cytokine function and alleviate inflammation-related diseases.
  • Atherosclerosis is a chronic inflammatory disease.
  • the main cause is abnormal lipid metabolism and accumulation on the vascular endothelium, triggering inflammation, aggravating the formation of plaque and thrombus, and leading to vascular blockage.
  • Inflammation always runs through all stages of the occurrence, development and evolution of atherosclerosis, and it may be the core link of the pathogenic mechanism of various inducing factors of atherosclerosis.
  • the inflammatory response induces the aggregation of M1-type polarized macrophages, which play the function of cleaning up accumulated lipids and necrotic cells, and at the same time release pro-inflammatory factors to accelerate the development of inflammation and plaque formation.
  • M2-type polarized macrophages are activated and release anti-inflammatory factors, promote plaque decomposition and exert anti-inflammatory repair function.
  • activation of M1-type polarized macrophages or inhibition of M2-type polarized macrophages can accelerate the formation of atherosclerotic plaques; on the contrary, activation of M2-type polarized macrophages can accelerate the formation of atherosclerotic plaques.
  • Macrophages can significantly inhibit the occurrence and development of atherosclerosis.
  • M1-type polarized macrophages were enriched in areas of severely inflamed plaques with lipid accumulation, while M2-type polarized macrophages were distributed in areas with a lower degree of inflammation.
  • clinical anti-inflammatory treatment strategies for atherosclerosis include the use of traditional anti-inflammatory drugs (such as statins, aspirin, etc.), inhibition of inflammatory-related factors TNF- ⁇ , interleukin activity (such as interleukin IL-1 ⁇ monoclonal antibody canakinumab) or inhibition of intracellular inflammatory signaling pathway-related molecules, such as inhibition of p38MAPK cascade kinase, NADPH oxidase and other inflammatory signaling molecules.
  • traditional anti-inflammatory drugs such as statins, aspirin, etc.
  • TNF- ⁇ interleukin activity
  • interleukin activity such as interleukin IL-1 ⁇ monoclonal antibody canakinumab
  • intracellular inflammatory signaling pathway-related molecules such as inhibition of p38MAPK cascade kinase, NADPH oxidase and other inflammatory signaling molecules.
  • many clinical drugs have the disadvantages of large side effects and weak specificity of inflammation inhibition.
  • regulating macrophage polarization and targeting the occurrence and development of inflammation have a key role in inhibiting the formation of atherosclerosis.
  • There are few methods for regulating the polarization of macrophages in the prior art and the inhibition of the occurrence and development of inflammation by regulating the polarization of macrophages still needs to be improved and developed. Regulate the occurrence and development of inflammation-related diseases.
  • the present application provides a method for regulating the polarization state of macrophages.
  • the cholinergic anti-inflammatory pathway is activated by drug stimulation or electrical stimulation of the vagus nerve, which affects the function of macrophages of different polarization types, thereby inhibiting the occurrence and development of inflammation. and inhibit the formation of atherosclerotic plaques.
  • the present application provides a method for modulating the polarization state of macrophages, the method comprising activating the cholinergic anti-inflammatory of macrophages using a cholinergic anti-inflammatory pathway agonist and/or a vagal electrical stimulation signal Pathway steps.
  • the polarization of macrophages promotes the polarization of M2 macrophages.
  • the cholinergic anti-inflammatory pathway agonist comprises acetylcholine and/or a cholinergic receptor agonist.
  • the cholinergic receptor agonist comprises GTS-21 (DMBX-A), which is a selective alpha7 nicotinic acetylcholine receptor agonist.
  • DMBX-A is a selective alpha7 nicotinic acetylcholine receptor agonist.
  • GTS-21 has the following structure:
  • acetylcholine or GTS-21 acts as a cholinergic anti-inflammatory pathway agonist, stimulates and activates the cholinergic anti-inflammatory pathway, inhibits the function of M1-type pro-inflammatory macrophages, and promotes M2-type anti-inflammatory
  • the function of macrophages has important applications in specifically regulating the function of polarized macrophages and the diseases they cause.
  • the concentration of the acetylcholine is 10-200 ⁇ M, for example, it can be 10 ⁇ M, 50 ⁇ M, 100 ⁇ M or 200 ⁇ M.
  • the concentration of the GTS-21 is 5-50 ⁇ M, such as 5 ⁇ M, 10 ⁇ M, 20 ⁇ M or 50 ⁇ M.
  • the applied voltage of the vagus nerve electrical stimulation signal is 3-6V, such as 3V, 4V, 5V or 6V, preferably 5V.
  • the application time of the vagus nerve electrical stimulation signal is 1 to 5 ms, for example, 1 ms, 2 ms, 3 ms, 4 ms or 5 ms, preferably 2 ms.
  • the application frequency of the vagus nerve electrical stimulation signal is 1 to 5 Hz, for example, 1 Hz, 2 Hz, 3 Hz, 4 Hz or 5 Hz, preferably 2 Hz.
  • the electrical stimulation signal stimulates the vagus nerve, activates the cholinergic anti-inflammatory pathway, inhibits the function of M1-type pro-inflammatory macrophages, and promotes the function of M2-type anti-inflammatory macrophages.
  • the macrophages comprise RAW264.7 and/or bone marrow-derived macrophages.
  • the present application provides a device for regulating the polarization state of macrophages, the device comprising a cholinergic anti-inflammatory pathway agonist applying unit and/or a vagus nerve electrical stimulation signal applying unit.
  • the cholinergic anti-inflammatory pathway agonist applying unit is used to apply acetylcholine and/or GTS-21 to macrophages.
  • the concentration of the acetylcholine is 10-200 ⁇ M, for example, it can be 10 ⁇ M, 50 ⁇ M, 100 ⁇ M or 200 ⁇ M.
  • the concentration of the GTS-21 is 5-50 ⁇ M, such as 5 ⁇ M, 10 ⁇ M, 20 ⁇ M or 50 ⁇ M.
  • the application time is 5-20 min, for example, it can be 5 min, 10 min, 15 min or 20 min.
  • the vagus nerve electrical stimulation signal applying unit is used for applying electrical stimulation signals to the vagus nerve.
  • the applied voltage of the electrical stimulation signal is 3-6V, such as 3V, 4V, 5V or 6V, preferably 5V.
  • the application time of the electrical stimulation signal is 1 to 5 ms, such as 1 ms, 2 ms, 3 ms, 4 ms or 5 ms, preferably 2 ms.
  • the application frequency of the electrical stimulation signal is 1 to 5 Hz, for example, 1 Hz, 2 Hz, 3 Hz, 4 Hz or 5 Hz, preferably 2 Hz.
  • the present application provides a pharmaceutical composition for regulating the polarization state of macrophages, the pharmaceutical composition comprising a cholinergic anti-inflammatory pathway agonist.
  • the cholinergic anti-inflammatory pathway agonist comprises acetylcholine and/or a cholinergic receptor agonist.
  • the cholinergic receptor agonist comprises GTS-21.
  • the pharmaceutical composition also includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
  • the pharmaceutical composition for activating the cholinergic anti-inflammatory pathway has a characteristic effect, and can be introduced into the body through intravenous injection, and transported to a specific site through blood circulation, so as to prevent and treat chronic cardiovascular diseases and gastrointestinal inflammatory diseases. disease, neurological disease or malignancy.
  • the present application provides the application of the device described in the second aspect and/or the pharmaceutical composition described in the third aspect in the preparation of a drug for treating inflammation-related diseases.
  • the disease comprises cardiovascular disease and/or neurological disease.
  • the disease comprises atherosclerosis.
  • the present application also provides a method for treating inflammation-related diseases, comprising:
  • a cholinergic anti-inflammatory pathway agonist is administered to the patient and/or a vagal electrical stimulation signal is applied to activate the cholinergic anti-inflammatory pathway of the patient's macrophages.
  • This application uses acetylcholine or GTS-21 to activate the cholinergic anti-inflammatory pathway, inhibit the function of M1-type pro-inflammatory macrophages, promote the function of M2-type anti-inflammatory macrophages, and achieve specific regulatory polarization Macrophage function and the effects of the diseases it causes;
  • the present application utilizes electrical stimulation of the vagus nerve to activate the cholinergic anti-inflammatory pathway, which is of great significance in the prevention and treatment of inflammatory related diseases.
  • Figure 1 is a technical solution roadmap
  • Figure 2 shows that acetylcholine and GTS-21 inhibit the release of pro-inflammatory factors from M1 macrophages
  • Figure 3 shows that acetylcholine and GTS-21 promote the polarization of M2 macrophages
  • Figure 4 shows that GTS-21-activated cholinergic anti-inflammatory pathway delays atherosclerotic inflammation and plaque formation after vagus nerve severing.
  • this application proposes a method for regulating the polarizing function of macrophages and delaying the occurrence and development of inflammation by using primary cell culture, histopathological observation and other technical means , mainly from the following aspects: 1) In vitro cell experiments confirmed the regulation of activation of cholinergic anti-inflammatory pathway on macrophage polarization; 2) Use of atherosclerosis mouse model to verify activation of cholinergic anti-inflammatory pathway The technical solution roadmap for the inhibitory effect on atherosclerotic inflammatory state and aortic plaque is shown in Figure 1.
  • mice were sacrificed by de-necking and immersed in two 75% alcohol beakers in turn; the thighs were cut with scissors, the femur and tibia were separated, the skin and flesh were removed, and then placed in 10 mL of RPMI1640 medium (containing 10% FBS); the femur and tibia were cut off.
  • RPMI1640 medium containing 10% FBS
  • the femur and tibia were cut off.
  • the bone marrow cavity is exposed, the bone marrow cavity is flushed with a 10mL syringe (1mL needle), and the cells are blown off with a 1mL pipette;
  • the cell suspension was collected into a 50 mL centrifuge tube; centrifuged at 1000 g for 8 min, the supernatant was discarded, and the cells were resuspended in 1640 medium containing 10 ng/mL macrophage colony-stimulating factor (MCSF), and seeded on 6 Cultured in a well plate; after three days, a medium containing 10 ng/mL MCSF was added, and mature bone marrow-derived macrophages (BMDM) were obtained after another 3 days of culture.
  • MCSF macrophage colony-stimulating factor
  • RAW264.7 or BMDM were inoculated in 6-well plates, and when the cell density reached about 70%, different concentrations of acetylcholine (0, 10, 50, 100, 200 ⁇ M) or cholinergic receptor agonist GTS-21 were added. (0, 5, 10, 20, 50 ⁇ M) for 10 min;
  • M1 macrophages were stimulated and induced with lipopolysaccharide LPS or IFN ⁇ , and the cells were collected after 24 h to analyze the expression of pro-inflammatory factors TNF ⁇ , IL-1 ⁇ and IL-6.
  • RAW264.7 or BMDM were inoculated in 6-well plates, and when the cell density reached about 70%, acetylcholine or cholinergic receptor agonist GTS-21 was added for 10 min;
  • M2 macrophages were stimulated and induced with IL-4, and the cells were collected after 24 h to analyze the expression of anti-inflammatory factors arginase 1 (Arg1), TGF- ⁇ and IL-10.
  • acetylcholine (Ach) and GTS-21 can significantly promote the differentiation of M2 macrophages and promote related Expression of marker genes such as Arg1, TGF and IL-10.
  • Apoe-/- mice were fed with high-fat diet (containing 21% fat, 0.15% cholesterol) for 10 weeks to construct atherosclerosis model mice; unilateral vagus nerve amputation;
  • mice were intraperitoneally injected with 4 mg/kg GTS-21, once every 3 days, to treat Apoe-/- mice on a high-fat diet;
  • a blank control group was set at the same time, and the same amount of normal saline was injected.
  • vagotomy significantly promoted the production of serum inflammatory factors TNFa and IL-6, and promoted the formation of arterial plaques;
  • GTS-21 treatment after vagus nerve cutting could significantly inhibit serum inflammatory factors TNFa and IL-6 and slow the formation of atherosclerotic plaques.
  • vagus nerve Using electrical stimulation signals (5V, 2ms, 2Hz) to stimulate the vagus nerve of atherosclerosis model mice, it was found that the expressions of serum pro-inflammatory factors TNFa, IL-1b and IL-6 were inhibited, while the anti-inflammatory factors TGF and IL-10
  • the increase in the expression of vagus nerve indicated that electrical stimulation of the vagus nerve also achieved regulation of the polarization state of macrophages, that is, inhibited the polarization of M1 macrophages, promoted the differentiation of M2 macrophages, and slowed down atherosclerotic plaques. Formation of blocks.
  • the present application activates the cholinergic anti-inflammatory pathway through drug stimulation or electrical stimulation of the vagus nerve, affecting the function of macrophages of different polarization types, thereby inhibiting the occurrence and development of inflammation, and is useful in the prevention and treatment of inflammatory diseases. significant.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

一种调节巨噬细胞极化状态的方法，所述方法包括采用胆碱能抗炎通路激动剂和/或迷走神经电刺激信号激活巨噬细胞的胆碱能抗炎通路的步骤。采用胆碱能抗炎通路激动剂对巨噬细胞进行体外刺激，或采用电刺激信号对迷走神经进行体内刺激，激活胆碱能抗炎通路，抑制了M1型巨噬细胞的极化，促进了M2型巨噬细胞的极化，实现了对巨噬细胞极化类型的调节作用，在特异性调节极化巨噬细胞功能及其引发的疾病方面具有重要意义。

Description

一种调节巨噬细胞极化状态的方法 技术领域
本申请属于生物医药技术领域，涉及一种调节巨噬细胞极化状态的方法，尤其涉及通过激活胆碱能抗炎通路调节巨噬细胞极化状态的方法及其在延缓动脉粥样硬化炎症发生发展中的应用。
背景技术
巨噬细胞是由骨髓系祖细胞分化为单核细胞后进入血液，迁移至组织中分化形成的。根据激活方式以及功能的差异，巨噬细胞分为经典型(M1型)和替代型(M2型)。在细菌毒素LPS或Th1类细胞因子(TNFα，IFNγ)等刺激下，巨噬细胞被激活极化为M1型，表达和释放促炎性因子和活性氧，具有杀灭病原体、清除坏死组织和促进炎症反应发生的作用；与之对应，在Th2类细胞因子(IL-4，IL-13)等诱导下，巨噬细胞被激活为M2型，表达抗炎因子(如TGFβ，IL-10)，具有消退炎症、促进组织生长和修复的作用。此外，通过改变Krüppel-like factors(KLF4)、STAT6、miR-33等基因的表达，也能调节巨噬细胞的极化状态。
研究发现，两种不同的极化巨噬细胞在炎症疾病的发生过程中具有重要的调控作用，在不同的微环境响应不同刺激时，可以在功能上相互转变；而阻断M1型巨噬细胞的功能，维持M2型极化细胞的抗炎修复特性，对抑制炎症的发生发展具有重要作用。因此，寻找一种有效的方法调节巨噬细胞的极化状态，在预防和治疗炎症相关疾病方面意义重大。然而，目前有效的调节巨噬细胞极化的方法较少。
胆碱能抗炎通路(Cholinergic Anti-inflammatory Pathway，CAP)是一种新近发现的神经免疫调节抗炎途径。在炎症条件下，传入迷走神经(afferent vagus)将炎症信号传递至中枢神经系统，再通过传出迷走神经(efferent vagus)释放乙 酰胆碱(Acetylcholine，ACh)作用于巨噬细胞，进而降低细胞炎症因子的释放，可以有效抑制炎症反应的发展。CAP对全身和局部炎症均具有明显的抑制作用，为调节细胞因子功能和缓解炎症相关疾病提供了有效途径。研究表明，通过刺激迷走神经激活CAP，能够抑制急性炎症、脓毒症、慢性类风湿性关节炎等疾病的发生发展，从而改善疾病症状。目前，刺激迷走神经的方法已被美国FDA认证通过，被应用于癫痫、类风湿性关节炎等疾病的预防和治疗。
动脉粥样硬化是一种慢性炎症性疾病，主要病因是脂质代谢异常并积累于血管内皮上，引发炎症，加剧斑块和血栓的形成，导致血管堵塞。炎症反应始终贯穿于动脉粥样硬化的发生、发展和演变的各个阶段，可能是动脉粥样硬化多种诱发因素致病机制的核心环节。在动脉粥样硬化发生初期，炎症反应诱导M1型极化巨噬细胞聚集，发挥清理累积的脂质及坏死细胞的功能，同时释放促炎症因子加快炎症发展以及斑块形成；而伴随炎症的加剧，M2型极化巨噬细胞被激活并释放抗炎因子，促进斑块分解并发挥抗炎修复功能。在对动脉粥样硬化动物模型的研究中发现，激活M1型极化巨噬细胞或者抑制M2型极化巨噬细胞，均可以加速动脉粥样硬化斑块的形成；相反，激活M2型极化巨噬细胞则可以显著抑制动脉粥样硬化的发生发展。对临床动脉粥样硬化病人组织切片分析发现，M1型极化巨噬细胞富集于脂质累积的重度炎症斑块区域，而M2型极化巨噬细胞则分布于炎症程度较低的区域。
目前，临床上针对动脉粥样硬化的抗炎治疗策略包括使用传统抗炎药物(如他汀类、阿司匹林等)、抑制炎症相关因子TNF-α、白介素类活性(如白介素IL-1β的单克隆抗体canakinumab)或抑制细胞内炎症信号通路相关分子，如抑制p38MAPK级联激酶、NADPH氧化酶等炎症信号分子活性。然而，临床药物多存在副作用大、炎症抑制特异性弱的缺点。
因此，调节巨噬细胞极化，靶向炎症的发生发展，对抑制动脉粥样硬化的形成具有关键作用。现有技术针对调节巨噬细胞极化的方法较少，而通过调节巨噬细胞极化影响抑制炎症发生发展还有待于改进和发展，需研发更加特异、效果更好的靶向调节方法，进而调节炎症相关疾病的发生发展。
发明内容
本申请提供了一种调节巨噬细胞极化状态的方法，通过药物刺激或者电刺激迷走神经激活胆碱能抗炎通路，影响不同极化类型的巨噬细胞的功能，从而抑制炎症的发生发展，并抑制动脉粥样硬化斑块的形成。
第一方面，本申请提供了一种调节巨噬细胞极化状态的方法，所述方法包括采用胆碱能抗炎通路激动剂和/或迷走神经电刺激信号激活巨噬细胞的胆碱能抗炎通路的步骤。
本申请中，采用胆碱能抗炎通路激动剂对巨噬细胞进行体外刺激，或采用电刺激信号对迷走神经进行体内刺激，均实现了对胆碱能抗炎通路的激活效果，抑制了M1型巨噬细胞的极化，促进了M2型巨噬细胞的极化。
优选地，所述胆碱能抗炎通路激动剂包括乙酰胆碱和/或胆碱受体激动药。
优选地，所述胆碱受体激动药包括GTS-21(DMBX-A)，是一种选择性的α7烟碱乙酰胆碱受体激动剂。
GTS-21具有如下结构：
Figure PCTCN2020124288-appb-000001
本申请中，乙酰胆碱或GTS-21作为胆碱能抗炎通路激动剂，刺激并激活胆碱能抗炎通路，抑制了M1型促炎性巨噬细胞的功能，并促进了M2型抑炎性巨 噬细胞的功能，在特异性调节极化巨噬细胞功能及其引发的疾病方面具有重要应用。
优选地，所述乙酰胆碱的浓度为10～200μM，例如可以是10μM、50μM、100μM或200μM。
优选地，所述GTS-21的浓度为5～50μM，例如可以是5μM、10μM、20μM或50μM。
优选地，所述迷走神经电刺激信号的施加电压为3～6V，例如可以是3V、4V、5V或6V，优选为5V。
优选地，所述迷走神经电刺激信号的施加时间为1～5ms，例如可以是1ms、2ms、3ms、4ms或5ms，优选为2ms。
优选地，所述迷走神经电刺激信号的施加频率为1～5Hz，例如可以是1Hz、2Hz、3Hz、4Hz或5Hz，优选为2Hz。
本申请中，电刺激信号刺激迷走神经，激活胆碱能抗炎通路，抑制了M1型促炎性巨噬细胞的功能，并促进了M2型抑炎性巨噬细胞的功能。
优选地，所述巨噬细胞包括RAW264.7和/或骨髓来源巨噬细胞。
第二方面，本申请提供了一种调节巨噬细胞极化状态的装置，所述装置包括胆碱能抗炎通路激动剂施加单元和/或迷走神经电刺激信号施加单元。
优选地，所述胆碱能抗炎通路激动剂施加单元用于向巨噬细胞施加乙酰胆碱和/或GTS-21。
优选地，所述乙酰胆碱的浓度为10～200μM，例如可以是10μM、50μM、100μM或200μM。
优选地，所述GTS-21的浓度为5～50μM，例如可以是5μM、10μM、20μM或50μM。
优选地，所述施加的时间为5～20min，例如可以是5min、10min、15min或20min。
优选地，所述迷走神经电刺激信号施加单元用于向迷走神经施加电刺激信号。
优选地，所述电刺激信号的施加电压为3～6V，例如可以是3V、4V、5V或6V，优选为5V。
优选地，所述电刺激信号的施加时间为1～5ms，例如可以是1ms、2ms、3ms、4ms或5ms，优选为2ms。
优选地，所述电刺激信号的施加频率为1～5Hz，例如可以是1Hz、2Hz、3Hz、4Hz或5Hz，优选为2Hz。
第三方面，本申请提供了一种调节巨噬细胞极化状态的药物组合物，所述药物组合物包括胆碱能抗炎通路激动剂。
优选地，所述胆碱能抗炎通路激动剂包括乙酰胆碱和/或胆碱受体激动药。
优选地，所述胆碱受体激动药包括GTS-21。
所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
本申请中，激活胆碱能抗炎通路的药物组合物具有特性作用性，可通过静脉注射导入生物体内，通过血液循环运输到特定部位，达到预防和治疗心血管慢性疾病、胃肠道炎症性疾病、神经系统疾病或恶性肿瘤的目的。
第四方面，本申请提供了第二方面所述的装置和/或第三方面所述的药物组合物在制备炎症相关疾病治疗药物中的应用。
优选地，所述疾病包括心血管疾病和/或神经系统疾病。
优选地，所述疾病包括动脉粥样硬化。
另一方面，本申请还提供了一种治疗炎症相关疾病的方法，包括：
向患者施用胆碱能抗炎通路激动剂和/或施加迷走神经电刺激信号，以激活患者的巨噬细胞的胆碱能抗炎通路。
与现有技术相比，本申请具有如下有益效果：
(1)本申请利用乙酰胆碱或GTS-21激活胆碱能抗炎通路，抑制M1型促炎性巨噬细胞的功能，促进M2型抑炎性巨噬细胞的功能，实现了特异性调节极化巨噬细胞功能及其引发的疾病的效果；
(2)本申请利用电刺激迷走神经激活胆碱能抗炎通路，在预防和治疗炎症性相关疾病方面具有重要意义。
附图说明
图1为技术方案路线图；
图2为乙酰胆碱和GTS-21抑制M1型巨噬细胞释放促炎性因子；
图3为乙酰胆碱和GTS-21促进M2型巨噬细胞极化；
图4为切断迷走神经后，GTS-21激活的胆碱能抗炎通路延缓动脉粥样硬化的炎症和斑块形成。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本申请从调节极化巨噬细胞功能、特异性干预炎症反应的角度出发，运用 原代细胞培养、组织病理观察等技术手段，提出一种调节巨噬细胞极化功能、延缓炎症发生发展的方法，主要从以下方面列举实施例：1)体外细胞实验证实激活胆碱能抗炎通路对巨噬细胞极化的调节作用；2)利用动脉粥样硬化小鼠模型验证胆碱能抗炎通路激活对动脉粥样硬化炎症状态和主动脉斑块的抑制作用，技术方案路线图如图1所示。
实施例1骨髓来源的巨噬细胞的分离和培养
将小鼠脱颈处死，依次浸泡于两个75％酒精烧杯中；用剪刀剪断大腿，分离股骨和胫骨，剔除皮肉后置于10mL RPMI1640培养基(含10％FBS)中；剪断股骨和胫骨的关节面，暴露骨髓腔，用10mL注射器(1mL针头)冲洗骨髓腔，1mL移液枪吹散细胞；
细胞经细胞筛过滤后，收集细胞悬浮液至50mL离心管中；1000g离心8min，弃上清，用含有10ng/mL巨噬细胞集落刺激因子(MCSF)的1640培养基重悬细胞，接种于6孔板中进行培养；三天后添加含有10ng/mL MCSF的培养基，再培养3天后获得的成熟骨髓来源的巨噬细胞(BMDM)。
实施例2乙酰胆碱和胆碱能受体激动剂对M1型巨噬细胞的影响
将RAW264.7或BMDM分别接种于6孔板中，待细胞密度达70％左右时，加入不同浓度的乙酰胆碱(0、10、50、100、200μM)或胆碱能受体激动剂GTS-21(0、5、10、20、50μM)处理10min；
使用脂多糖LPS或IFNγ刺激诱导M1巨噬细胞，24h后收集细胞，分析促炎性因子TNFα、IL-1β、IL-6的表达。
结果如图2所示，在LPS诱导的RAW264.7细胞中，分别用不同浓度乙酰胆碱(Ach)和GTS-21进行处理24小时，然后分析促炎性因子TNFa、IL-1b和IL-6的相对表达，结果显示，LPS诱导产生的促炎性因子能够被乙酰胆碱和 GTS-21抑制，并呈现浓度依赖性。与此类似，在IFNg诱导的原代骨源性巨噬细胞中，乙酰胆碱(Ach)和GTS-21能够浓度依赖性的抑制促炎性因子TNFa、IL-1b和IL-6的增加。
实施例3乙酰胆碱和胆碱能受体激动剂对M2型巨噬细胞的影响
将RAW264.7或BMDM分别接种于6孔板中，待细胞密度达70％左右时，加入乙酰胆碱或胆碱能受体激动剂GTS-21处理10min；
使用IL-4刺激诱导M2巨噬细胞，24h后收集细胞，分析抗炎性因子精氨酸酶1(Arg1)、TGF-β、IL-10的表达。
结果如图3所示，在IL4诱导的M2型巨噬细胞RAW264.7和原代骨髓源巨噬细胞中，乙酰胆碱(Ach)和GTS-21能够明显促进M2型巨噬细胞的分化，促进相关标志基因如Arg1、TGF以及IL-10的表达。
实施例4GTS-21或电刺激对Apoe-/-小鼠动脉粥样硬化的影响
以高脂饲料(含21％脂肪，0.15％胆固醇)喂养Apoe-/-小鼠10周，构建动脉粥样硬化模型小鼠；10周后，对小鼠颈部下行的、位于颈动脉鞘内的单侧迷走神经进行切断手术；
向小鼠腹腔注射4mg/kg GTS-21，每3天一次，处理高脂饲料Apoe-/-小鼠；
实验结束后，眼球取血分析血清炎症因子的表达，并分离主动脉进行血管斑块分析。
本实施例同时设置空白对照组，注射等量的生理盐水。
结果如图4所示，迷走神经切断明显促进了血清炎症因子TNFa和IL-6的产生，并促进动脉斑块的形成；而切断迷走神经后GTS-21处理能够明显抑制血清炎症因子TNFa和IL-6的产生，并减缓动脉粥样硬化斑块的形成。
采用电刺激信号(5V、2ms、2Hz)刺激动脉粥样硬化模型小鼠的迷走神 经，发现血清促炎症因子TNFa、IL-1b和IL-6的表达得到抑制，而抗炎因子TGF和IL-10的表达提高，说明电刺激迷走神经同样实现了对巨噬细胞极化状态的调节，即抑制了M1型巨噬细胞的极化，促进了M2型巨噬细胞的分化，减缓了动脉粥样硬化斑块的形成。
综上所述，本申请通过药物刺激或者电刺激迷走神经激活胆碱能抗炎通路，影响不同极化类型的巨噬细胞的功能，从而抑制炎症的发生发展，在炎症性疾病的预防和治疗方面具有重要意义。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种调节巨噬细胞极化状态的方法，其包括采用胆碱能抗炎通路激动剂和/或迷走神经电刺激信号激活巨噬细胞的胆碱能抗炎通路的步骤。
  2. 根据权利要求1所述的方法，其中，所述胆碱能抗炎通路激动剂包括乙酰胆碱和/或胆碱受体激动药。
  3. 根据权利要求2所述的方法，其中，所述胆碱受体激动药包括GTS-21。
  4. 根据权利要求2所述的方法，其中，所述乙酰胆碱的浓度为10～200μM。
  5. 根据权利要求3所述的方法，其中，所述GTS-21的浓度为5～50μM。
  6. 根据权利要求1-5中任一项所述的方法，其中，所述迷走神经电刺激信号的施加电压为3～6V；
    优选地，所述迷走神经电刺激信号的施加时间为1～5ms；
    优选地，所述迷走神经电刺激信号的施加频率为1～5Hz。
  7. 根据权利要求1-6任一项所述的方法，其中，所述巨噬细胞包括RAW264.7和/或骨髓来源巨噬细胞。
  8. 一种调节巨噬细胞极化状态的装置，其包括胆碱能抗炎通路激动剂施加单元和/或迷走神经电刺激信号施加单元。
  9. 根据权利要求8所述的装置，其中，所述胆碱能抗炎通路激动剂施加单元用于向巨噬细胞施加乙酰胆碱和/或GTS-21；
    优选地，所述乙酰胆碱的浓度为10～200μM；
    优选地，所述GTS-21的浓度为5～50μM；
    优选地，所述施加的时间为5～20min。
  10. 根据权利要求8所述的装置，其中，所述迷走神经电刺激信号施加单元用于向迷走神经施加电刺激信号；
    优选地，所述电刺激信号的施加电压为3～6V；
    优选地，所述电刺激信号的施加时间为1～5ms；
    优选地，所述电刺激信号的施加频率为1～5Hz。
  11. 一种调节巨噬细胞极化状态的药物组合物，其包括胆碱能抗炎通路激动剂；
    优选地，所述胆碱能抗炎通路激动剂包括乙酰胆碱和/或胆碱受体激动药；
    优选地，所述胆碱受体激动药包括GTS-21。
  12. 根据权利要求11所述的药物组合物，其中，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  13. 权利要求8-10任一项所述的装置和/或权利要求11或12所述的药物组合物在制备炎症相关疾病治疗药物中的应用。
  14. 根据权利要求13所述的应用，其中，所述疾病包括心血管疾病、脓血症、风湿性关节炎、神经系统疾病或肿瘤中的任意一种或至少两种的组合。
  15. 根据权利要求13所述的应用，其中，所述疾病包括动脉粥样硬化。
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