WO2022000705A1 - Egcg在制备预防病原细菌感染的制剂或食品中的应用 - Google Patents

Egcg在制备预防病原细菌感染的制剂或食品中的应用 Download PDF

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WO2022000705A1
WO2022000705A1 PCT/CN2020/107719 CN2020107719W WO2022000705A1 WO 2022000705 A1 WO2022000705 A1 WO 2022000705A1 CN 2020107719 W CN2020107719 W CN 2020107719W WO 2022000705 A1 WO2022000705 A1 WO 2022000705A1
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egcg
infection
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pathogenic bacteria
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卫林
韩晓阳
陈悦
杨洋
徐薇
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Suzhou University
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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the technical field of bacterial infection prevention, in particular to the application of EGCG in the preparation of preparations or foods for preventing pathogenic bacterial infection.
  • Tea polyphenols (the general term for all polyphenolic compounds in green tea) are the main water-soluble components in tea, and are considered to be important substances for green tea to exert physiological and pharmacological functions.
  • Botanicals Among tea polyphenols, catechins account for about 60-80%, of which, epigallocatechin gallate (EGCG) is the component with the highest content in catechins, accounting for about 60% of catechins. 50%-60% of class compounds.
  • EGCG epigallocatechin gallate
  • EGCG has a variety of biological activities, including: antibacterial, antiviral, anti-arteriosclerosis, anti-thrombosis, anti-vascular proliferation, anti-inflammatory, anti-tumor, scavenging free radicals, reducing oxidative damage, etc. But it is not toxic to normal tissues and cells.
  • EGCG has a broad-spectrum antibacterial function, which can inhibit the infection of Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Salmonella typhi, Shigella Shigella, Bacillus cholera, Pseudomonas aeruginosa, Bacillus subtilis, and oral Streptococcus mutans have inhibitory effects, indicating that EGCG has good application prospects in the research and development of anti-infective biomedicine and functional food.
  • Gram-positive and Gram-negative bacteria including Staphylococcus aureus, Salmonella typhi, Shigella Shigella, Bacillus cholera, Pseudomonas aeruginosa, Bacillus subtilis, and oral Streptococcus mutans have inhibitory effects, indicating that EGCG has good application prospects in the research and development of anti-infective biomedicine and functional food.
  • the purpose of the present invention is to provide a kind of application of EGCG in the preparation of preparations or foods for preventing pathogenic bacterial infection.
  • the present invention discloses the preventive function and action mechanism of EGCG on pathogenic bacterial infection, which is the anti-infection of EGCG. New directions for drug development.
  • the purpose of the present invention is to disclose the application of epigallocatechin gallate (EGCG) in the preparation of preparations or foods for preventing pathogenic bacterial infection.
  • EGCG epigallocatechin gallate
  • epigallocatechin gallate is derived from green tea.
  • pathogenic bacteria include Gram-positive bacteria and/or Gram-negative bacteria.
  • the pathogenic bacteria include one or more of Escherichia coli ATCC25922, Acinetobacter baumannii ATCC19606, Staphylococcus aureus ATCC25923 and Methicillin-resistant Staphylococcus aureus MRSA.
  • the infection site of the pathogenic bacteria is the abdominal cavity.
  • the preparation is a medicine
  • the medicine is an injection.
  • the single dose of the injection is 1-10 mg/kg/day.
  • the food is health food or beverage.
  • epigallocatechin gallate is used to reduce bacterial load at the site of infection.
  • epigallocatechin gallate is used to promote chemotactic migration of neutrophils, monocytes and macrophages to the site of infection.
  • epigallocatechin gallate induces the production of cytokines and chemokines in mouse peritoneal macrophages by activating macrophage innate immunity-related cell signaling pathways.
  • EGCG induces mouse peritoneal macrophages to produce certain levels of cytokines and chemokines by activating the phosphorylation of p38 and JNK in mouse peritoneal macrophages.
  • the present invention has at least the following advantages:
  • the present invention clarifies that the main active component of green tea---EGCG can effectively enhance the "immunity" of the body from the perspective of EGCG's regulation of the body's innate immune pattern, thereby reducing the risk of the body being infected by pathogenic bacteria, and EGCG is effective against pathogenic bacteria infection.
  • the preventive effect of EGCG is significantly better than the therapeutic effect, which provides a new direction for the research and development of anti-infective drugs of EGCG.
  • Figure 1 is the bacterial load test results of different experimental groups after infection of the abdominal cavity with different pathogenic bacteria
  • Figure 2 is a graph showing the percentage of neutrophils, monocytes and macrophages in the abdominal cavity after intraperitoneal injection of EGCG and the statistical results of the absolute number of cells;
  • Fig. 3 is the test result of cytokine and chemokine levels in the local abdominal cavity after intraperitoneal injection of EGCG;
  • Figure 4 is (A) Western blot and (B) gray value statistics of the phosphorylation of p38 and JNK in EGCG-activated mouse peritoneal macrophages;
  • ns means no significant difference, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • mice can significantly enhance the prevention of pathogenic bacterial infection in mice
  • mice C57BL/6 mice (18-22 g, purchased from Shanghai Slack) were randomly divided into 6 mice in each group.
  • mice were injected intraperitoneally with EGCG (10 mg/kg) 24h (-24h), or 8h (-8h) before bacterial infection.
  • mice were intraperitoneally injected with EGCG (10 mg/kg) at 0h (0h) and 4h (+4h) after bacterial infection.
  • negative control mice PBS were given an equal volume of PBS.
  • mice in the above groups were injected intraperitoneally with Escherichia coli ATCC25922, Acinetobacter baumannii ATCC19606, Staphylococcus aureus ATCC25923 or methicillin-resistant Staphylococcus aureus ( MRSA), the injection dose of bacteria is about 2.2 ⁇ 10 7 CFU/only.
  • Ctrl (control) was a normal group of mice without any injection of drugs or bacteria. 18 hours after the mice in each group were injected with bacteria, the mice were sacrificed with carbon dioxide, and 2 mL of ice-cold PBS was used to flush the abdominal cavity of the mice with a syringe, and ascites was collected.
  • Bacterial CFU Bacterial CFU.
  • mice injected with EGCG at -24h and -8h were significantly lower than that of mice injected with EGCG at +4h, indicating that the preventive effect of EGCG on pathogenic bacterial infection was better than the therapeutic effect.
  • Intraperitoneal injection of EGCG promotes chemotactic migration of neutrophils and monocytes/macrophages to the local peritoneal cavity of mice
  • Neutrophils and monocytes/macrophages infiltrating early in local bacterial infection are important phagocytic bactericidal cells that play a crucial role in clearing pathogenic bacteria.
  • the present invention injects EGCG (10 mg/kg) into the abdominal cavity of mice, and sacrifices the mice with carbon dioxide 4h, 8h and 24h after injection, and sucks 2mL with a syringe.
  • mice Rinse the peritoneal cavity of mice with PBS, collect ascites, centrifuge at 1000g for 10 minutes, collect peritoneal cells, take about 1 ⁇ 10 6 cells and resuspend them in EP tube with PBS (100 ⁇ L), add cell surface marker antibodies, including CD45, CD11B, Ly6G, Ly6C and F4/80 were incubated at 4°C in the dark for 30 min, then centrifuged at 1000g for 10 min, the supernatant was discarded, washed with PBS, resuspended in 300 ⁇ L of PBS, and detected by flow cytometry (BD FACSCanto II).
  • cell surface marker antibodies including CD45, CD11B, Ly6G, Ly6C and F4/80 were incubated at 4°C in the dark for 30 min, then centrifuged at 1000g for 10 min, the supernatant was discarded, washed with PBS, resuspended in 300 ⁇ L of PBS, and detected by flow cytometry
  • EGCG can induce the production of cytokines and chemokines in mouse peritoneal macrophages
  • the present invention examines the effect of EGCG on the production of cytokines and chemokines by macrophages in mouse peritoneal cavity.
  • the mouse peritoneal macrophages were taken and plated on a 24-well cell culture plate (about 1 ⁇ 10 5 /well), EGCG (10, 20 and 40 ⁇ g/mL) was added, and the control group (Ctrl) was added with an equal volume of PBS, and cultured.
  • cytokines TNF- ⁇ , IL-1 ⁇ and IL-6
  • chemokines CXCL1, CXCL2 and MCP-1
  • EGCG significantly promoted the production of certain levels of cytokines (TNF- ⁇ and IL-1 ⁇ ) and chemokines (CXCL1, CXCL2 and MCP) in macrophages in a dose-dependent manner. -1). It was shown that EGCG can induce mouse peritoneal macrophages to produce certain levels of cytokines (TNF- ⁇ , IL-1 ⁇ and IL-6) and chemokines (CXCL1, CXCL2 and MCP-1), and then promote neutrophils, Monocytes/macrophages are recruited locally to the peritoneal cavity.
  • EGCG can activate innate immunity-related signaling pathways in mouse peritoneal macrophages
  • mouse peritoneal macrophages were taken and plated on 6-well cell culture plates (about 1 ⁇ 10 6 /well). ), EGCG (10, 20 and 40 ⁇ g/mL) was added, an equal volume of PBS was added to the negative control group (Ctrl), and LPS (100 ng/mL) was used as a positive control.
  • Protein samples were prepared, electrophoresed by SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat milk powder at room temperature for 2 hours, added with primary antibody overnight, washed with PBST, and incubated with secondary antibody at room temperature for 1 hour, washed with PBST, and then added with luminescent substrate. Expose, develop and fix.
  • EGCG can significantly activate the MAPKs signaling pathway, including activation of p38 and JNK phosphorylation.
  • the results showed that EGCG may induce mouse peritoneal macrophages to produce certain levels of cytokines and chemokines by activating the phosphorylation of p38 and JNK in mouse peritoneal macrophages.
  • the above results of the present invention reveal that EGCG has a preventive function against pathogenic bacterial infection, clarify the mechanism of EGCG preventing pathogenic bacterial infection, and provide a new direction for the research and development of anti-infective drugs of EGCG.
  • the results of the present invention provide a theoretical basis for the health function of green tea and the research and development of green tea-related health care products, suggest that drinking green tea may have the effect of preventing pathogenic bacterial infection, and expand people's understanding of the health and pharmacological functions of green tea. and understanding of its mechanism.

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Abstract

涉及表没食子儿茶素没食子酸酯(EGCG)在制备预防病原细菌感染的制剂或食品中的应用。并公开了EGCG对病原细菌感染的预防功能和作用机制，为EGCG的抗感染药物研发提供新方向。

Description

EGCG在制备预防病原细菌感染的制剂或食品中的应用 技术领域
本发明涉及细菌感染预防技术领域，尤其涉及一种EGCG在制备预防病原细菌感染的制剂或食品中的应用。
背景技术
长期以来，因绿茶中富含多种天然活性物质，绿茶的生理和药理功能一直备受人们的关注。茶多酚(绿茶中所有多酚类化合物的总称)是茶叶中主要的水溶性成分，被认为是绿茶发挥生理和药理功能的重要物质，也是第一个被美国食品药品监督管理局批准上市的植物药。在茶多酚中，儿茶素类化合物约占60-80％，其中，表没食子儿茶素没食子酸酯(Epigallocatechin gallate，EGCG)是儿茶素中含量最高的组分，约占儿茶素类化合物的50％-60％。已有的研究表明，EGCG具有多种生物学活性，具体包括：抗菌、抗病毒、抗动脉硬化、抗血栓形成、抗血管增生、抗炎、抗肿瘤、清除自由基、降低氧化损伤等作用，但对正常的组织和细胞没有毒性。
目前，诸多实验显示，EGCG具有广谱的抗菌功能，可以抑制革兰氏阳性菌、革兰氏阴性菌的感染，包括金黄色葡萄球菌、伤寒沙门氏杆菌、志贺氏痢疾杆菌、霍乱杆菌、铜绿色假单孢杆菌、枯草杆菌、口腔变异链球菌等均有抑制作用，表明EGCG在抗感染生物医药和功能食品研发方面，具有良好的应用前景。总结下来，目前人们发现的EGCG抑制细菌感染的主要机制有：(1)作用于细菌的细胞膜，EGCG可以结合细菌细胞膜，破坏菌体的壁膜结构，进而抑制细菌的生长；(2)作用于细菌形成的生物膜，细菌形成的生物膜是导致慢性感染的关键因素之一，近期研究表明EGCG可以抑制细菌生物膜的形成，进而抑制细菌的感染；(3)作用于细菌的耐药蛋白，金黄色葡萄球菌的甲氧西林耐药主要由mecA基因介导，该基因编码青霉素结合蛋白2a(PBP2a)，而EGCG能够抑制PBP2a的产生发挥抑菌作用。综上所述，目前科学家们主要聚焦在EGCG对病原细菌感染的治疗作用，目前的研究表明EGCG对细菌或细菌致病因子具有直接的抑制作用，从而抑制细菌的感染。
然而，EGCG作为人们日常饮用绿茶的主要活性成分，是否具有预防病原细菌感染的功能呢？已有的研究尚未进行阐述。
论文“表没食子儿茶素没食子酸酯对大肠杆菌引起的膀胱上皮感染预防和治疗作用的研究，刘述成”中公开了EGCG对大肠杆菌引起的膀胱上皮感染的预防作用，但是该论文中的 预防细菌感染作用是依靠抑制大肠杆菌感染导致的膀胱上皮细胞损伤和抑制大肠杆菌感染导致的炎症反应实现的，并未揭示EGCG通过对机体本底免疫的调控，进而实现预防病原细菌感染的功能，也没有揭示EGCG的提前干预对细菌载量的影响。
发明内容
为解决上述技术问题，本发明的目的是提供一种EGCG在制备预防病原细菌感染的制剂或食品中的应用，本发明公开了EGCG对病原细菌感染的预防功能和作用机制，为EGCG的抗感染药物研发提供新方向。
本发明的目的是公开表没食子儿茶素没食子酸酯(EGCG)在制备预防病原细菌感染的制剂或食品中的应用。
进一步地，表没食子儿茶素没食子酸酯来源于绿茶。
进一步地，病原细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
进一步地，病原细菌包括大肠杆菌Escherichia coli ATCC25922、鲍曼不动杆菌Acinetobacter baumanniiATCC19606、金黄色葡萄球菌Staphylococcus aureus ATCC25923和耐甲氧西林金黄色葡萄球菌MRSA中的一种或几种。
进一步地，病原细菌感染部位为腹腔。
进一步地，制剂为药物，所述药物为注射剂。
进一步地，注射剂的单剂量为1-10mg/kg/天。
进一步地，食品为保健食品或饮料。
进一步地，表没食子儿茶素没食子酸酯用于降低感染部位细菌载量。
进一步地，表没食子儿茶素没食子酸酯用于促进中性粒细胞、单核细胞和巨噬细胞趋化迁移至感染部位。
进一步地，表没食子儿茶素没食子酸酯通过激活巨噬细胞固有免疫相关细胞信号通路而诱导小鼠腹腔巨噬细胞产生细胞因子和趋化因子。
进一步地，EGCG通过激活小鼠腹腔巨噬细胞p38和JNK的磷酸化，进而诱导小鼠腹腔巨噬细胞产生一定水平的细胞因子和趋化因子。
借由上述方案，本发明至少具有以下优点：
本发明从EGCG对机体固有免疫格局调控的角度，阐明了绿茶主要活性成分---EGCG可以有效增强机体的“免疫力”，从而降低了机体被病原细菌感染的风险，且EGCG对病原细菌感染的预防效果显著优于治疗效果，为EGCG的抗感染药物研发提供新方向。
此外，从药食同源的角度，提示了EGCG在开发预防病原细菌感染的食品中的应用，拓展了人们对含EGCG的产品(如绿茶等)的健康功能和药理功能及其机制的认识，为含EGCG的产品的健康功能及绿茶相关的保健产品的研发提供理论依据。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是不同实验组在不同病原细菌的感染腹腔后的细菌载量测试结果；
图2是腹腔注射EGCG后腹腔局部的中性粒细、单核细胞、巨噬细胞的细胞比例图好细胞绝对数图统计结果；
图3是腹腔注射EGCG后腹腔局部的细胞因子和趋化因子水平测试结果；
图4为EGCG激活小鼠腹腔巨噬细胞的p38和JNK的磷酸化的(A)Westernblot图和(B)灰度值统计图；
图1-4中，ns表示没有显著差异，*p<0.05,**p<0.01,***p<0.001。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
EGCG可以显著增强小鼠预防病原细菌的感染
C57BL/6小鼠(18-22g，购买自上海斯莱克)随机分组，每组6只。为了检测EGCG对细菌感染的预防作用，在细菌感染前24h(-24h)、或8h(-8h)给小鼠腹腔注射EGCG(10mg/kg)。同时为了作为对照，比较EGCG对细菌感染的治疗作用和预防作用的效果，在细菌感染后0h(0h)、4h(+4h)给小鼠腹腔注射EGCG(10mg/kg)。另外，阴性对照组小鼠(PBS)给予等体积PBS。以上组别的小鼠均在第0h分别腹腔注射大肠杆菌(Escherichia coli ATCC25922)、鲍曼不动杆菌(Acinetobacter baumanniiATCC19606)、金黄色葡萄球菌(Staphylococcus aureus ATCC25923)或耐甲氧西林金黄色葡萄球菌(MRSA)，细菌的注射剂量约为2.2×10 7CFU/只。此外，Ctrl(control)为正常组小鼠，不注射任何药物或细菌。各组小鼠注射细菌18h后，用二氧化碳处死小鼠，用注射器吸取2mL冰上预冷的PBS冲洗小鼠腹腔，并收集腹水，取50μL涂布于LB平板上，37℃倒置培养过夜，计数细菌的CFU。
结果如图1所示，结果表明，与PBS组相比，预注射EGCG的小鼠的细菌载量显著降 低，-24h和-8h注射EGCG均显著减少了小鼠腹腔的大肠杆菌(图1a)、鲍曼不动杆菌(图1b)、金黄色葡萄球菌(图1c)和耐甲氧西林金黄色葡萄球菌(图1d)的细菌载量，表明EGCG可以显著预防病原细菌的感染；此外，我们发现-24h和-8h注射EGCG的小鼠腹腔细菌载量显著低于+4h注射EGCG的小鼠，表明EGCG对病原细菌感染的预防效果优于治疗效果。
实施例2
腹腔注射EGCG可促进中性粒细胞、单核/巨噬细胞趋化迁移至小鼠腹腔局部
细菌感染局部早期浸润的中性粒细胞和单核/巨噬细胞是重要的吞噬杀菌细胞，对清除病原细菌起着至关重要的作用。为了明确EGCG是否可以招募吞噬细胞，进而促进小鼠清除病原细菌，本发明给小鼠腹腔注射EGCG(10mg/kg)，分别在注射4h、8h和24h后用二氧化碳处死小鼠，用注射器吸取2mL的PBS冲洗小鼠腹腔，收集腹水，1000g离心10分钟，收集腹腔细胞，取约1×10 6个细胞用PBS(100μL)重悬于EP管中，加入细胞表面标记抗体，包括CD45，CD11B，Ly6G，Ly6C和F4/80，4℃避光孵育30min，再1000g离心10min，弃上清，PBS洗涤，用300μL的PBS重悬，用流式细胞仪(BD FACSCanto Ⅱ)检测。
结果如图2所示，与对照组(Ctrl，给予等体积的PBS)相比，腹腔注射EGCG后4h、8h和24h，小鼠腹水中的中性粒细胞(Neutrophils，图2b1)、Ly6C high巨噬细胞(Ly6C highmacrophages，图2b2)、Ly6C high单核细胞(Ly6C highmonocytes，图2b3)的总数显著增加，表明EGCG可以显著促进对中性粒细胞、单核/巨噬细胞向腹腔局部招募。
实施例3
EGCG可以诱导小鼠腹腔巨噬细胞产生细胞因子和趋化因子
为了明确EGCG是如何招募中性粒、单核/巨噬细胞到腹腔局部，本发明检测了EGCG对小鼠腹腔巨噬细胞产生细胞因子和趋化因子的影响。取小鼠腹腔巨噬细胞，铺板于24孔细胞培养板上(约1×10 5/孔)，加入EGCG(10、20和40μg/mL)，对照组(Ctrl)加入等体积的PBS，培养4h后，用ELISA试剂盒(购买于联科生物)检测上清中的细胞因子(TNF-α、IL-1β和IL-6)和趋化因子(CXCL1、CXCL2和MCP-1)的水平。
结果如图3所示，与PBS处理组相比，EGCG以剂量依赖的方式显著促进巨噬细胞产生一定水平的细胞因子(TNF-α和IL-1β)和趋化因子(CXCL1、CXCL2和MCP-1)。表明EGCG可以诱导小鼠腹腔巨噬细胞产生一定水平的细胞因子(TNF-α、IL-1β和IL-6)和趋化因子(CXCL1、CXCL2和MCP-1)，进而促进中性粒细胞、单核/巨噬细胞招募至腹腔局部。
实施例4
EGCG可以激活小鼠腹腔巨噬细胞固有免疫相关信号通路
为了探索EGCG是否通过激活巨噬细胞固有免疫相关细胞信号通路进而诱导细胞因子和趋化因子的产生，取小鼠腹腔巨噬细胞，铺板于6孔细胞培养板上(约1×10 6/孔)，加入EGCG(10、20和40μg/mL)，阴性对照组(Ctrl)加入等体积的PBS，LPS(100ng/mL)作为阳性对照，37℃孵育45min后，用RIPA裂解液将细胞裂解，制备蛋白样品，用SDS-PAGE电泳后，转至PVDF膜上，5％脱脂奶粉室温封闭2h，加入一抗过夜，PBST洗涤后，加入二抗室温孵育1h，PBST洗涤后，加入发光底物进行曝光、显影和定影。
结果如图4所示，EGCG可以显著激活MAPKs信号通路，包括激活p38和JNK的磷酸化。结果表明，EGCG可能是通过激活小鼠腹腔巨噬细胞p38和JNK的磷酸化，进而诱导小鼠腹腔巨噬细胞产生一定水平的细胞因子和趋化因子。
综上，本发明以上结果揭示了EGCG对病原细菌感染具有预防功能，阐明了EGCG预防病原细菌感染的作用机制，为EGCG的抗感染药物研发提供新方向。同时由于EGCG来源于绿茶，本发明的结果为绿茶的健康功能和绿茶相关的保健品研发提供理论依据，提示了饮用绿茶可能具有预防病原细菌感染的功效，拓展了人们对绿茶健康功能和药理功能及其机制的认识。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (10)

  1. 表没食子儿茶素没食子酸酯在制备预防病原细菌感染的制剂或食品中的应用。
  2. 根据权利要求1所述的应用，其特征在于：病原细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
  3. 根据权利要求1或2所述的应用，其特征在于：所述病原细菌包括大肠杆菌Escherichia coli ATCC25922、鲍曼不动杆菌Acinetobacter baumanniiATCC 19606、金黄色葡萄球菌Staphylococcus aureus ATCC25923和耐甲氧西林金黄色葡萄球菌MRSA中的一种或几种。
  4. 根据权利要求1所述的应用，其特征在于：所述病原细菌感染部位为腹腔。
  5. 根据权利要求1所述的应用，其特征在于：所述制剂为药物，所述药物为注射剂。
  6. 根据权利要求5所述的应用，其特征在于：所述注射剂的单剂量为1-10mg/kg/天。
  7. 根据权利要求1所述的应用，其特征在于：所述食品为保健食品或饮料。
  8. 根据权利要求1所述的应用，其特征在于：所述表没食子儿茶素没食子酸酯用于降低感染部位细菌载量。
  9. 根据权利要求1所述的应用，其特征在于：所述表没食子儿茶素没食子酸酯用于促进中性粒细胞、单核细胞和巨噬细胞趋化迁移至感染部位。
  10. 根据权利要求9所述的应用，其特征在于：所述表没食子儿茶素没食子酸酯通过激活巨噬细胞固有免疫相关细胞信号通路而诱导小鼠腹腔巨噬细胞产生细胞因子和趋化因子。
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