炎症反应是免疫系统动员免疫细胞清除有害刺激的防御机制，根 据刺激物和效应细胞的不同，学界将引起炎症的免疫反应分为1~3型，其中以1型和2型免疫最为常见。1型免疫（Type 1 immunity）是指机体针对可进入细胞内的细菌、病毒等微生物感染所启动的免疫反应，而2型免疫（Type 2 immunity）则是机体为了清除寄生蠕虫或环境中有害物质而激活的免疫反应。无论是1型还是2型免疫均会引起各自不同特点的炎症反应。所以，1型免疫也常被称为1型炎症（Type 1 inflammation），而2型免疫则称为2型炎症（Type 2 inflammation）。炎症是机体清除有害刺激时所带来的必然反应，但如果炎症的程度或持续时间未得控制，免疫细胞持续活化，则会给机体带来损害（如细胞因子风暴、特应性疾病），甚至可危及生命（如败血症、过敏性休克）。因此，针对1型或2型炎症的抗炎药物既是临床之所需，也是新药研究的热点。

       桂花（O. fragrans flower）是木犀科植物木犀（Osmanthus fragrans (Thunb.) Lour.）的干燥花，桂花的香味细腻醇厚，且留香持久，是一种典型的药食两用花卉，自古被广泛添加到各类食品及饮料中。其中，桂花酒因其制作简单、功效显著而倍受欢迎。传统认为桂花具有化痰、止痛、消肿、止肠风血痢等功效，可用于治疗哮喘、牙痛、胃痛、腹泻等炎症相关疾病。现代研究发现，桂花富含黄酮、多酚、苯乙醇苷类等有效成分，具有突出的抗氧化、抗肿瘤、镇痛、抗糖尿病等作用，且毒性低。在抗炎方面，有研究显示桂花提取物能够减轻小鼠结肠炎和多种特应性疾病(过敏性气道炎、过敏性鼻炎、湿疹等)，提示桂花可能具有抗1型和/或2型炎症的作用。因此，本研究分别聚焦于1型炎症的主效应细胞（巨噬细胞）和2型炎症的主效应细胞（肥大细胞），对桂花乙醇提取物（ethanol extract of O. fragrans flower, OFE）对两种细胞活化的对抗作用及其分子机制展开了初步探索。

         在抗巨噬细胞活化方面，本实验探究了OFE对脂多糖（lipopolysaccharide, LPS）诱导巨噬细胞炎症反应的作用及机制。在小鼠内毒素血症模型上，单次腹腔注射OFE能够显著降低小鼠血清中促炎因子白介素-6（interleukin-6, IL-6）和单核细胞趋化因子-1（monocyte chemoattractant protein-1, MCP-1）水平。在LPS活化小鼠腹腔原代巨噬细胞和RAW264.7细胞模型上，OFE均能剂量依赖地显著减少促炎因子一氧化氮（nitric oxide, NO）、IL-6、MCP-1和白介素-1β（interleukin-1β, IL-1β）的生成。机制研究结果表明，OFE并不影响诱导型NO合成酶（inducible NO synthase, iNOS）的活性，而是通过抑制该酶的基因转录和翻译来减少NO的生成；OFE也能显著抑制IL-6、MCP-1和IL-1β的基因转录。由于上述促炎因子的基因转录受到转录因子NF-κB和AP-1调控，本研究接下来分别考察了OFE对这两种转录因子活化的影响。结果显示，OFE对NF-κB和AP-1的活化均具有显著抑制作用：它能减少NF-κB抑制蛋白IκBα的磷酸化水平并阻止其降解、以及随后的NF-κB核易位，最终抑制NF-κB信号；同时，OFE还能够降低AP-1上游丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPKs）家族成员ERK、p38、JNK的磷酸化水平，从而抑制AP-1信号活化。

        在抗肥大细胞活化方面，本研究初步探索了OFE对IgE依赖型和IgE非依赖型肥大细胞炎症反应的作用及机制：（1）IgE-FcεRI复合物与特异性抗原交联后启动IgE依赖型肥大细胞活化，本研究利用鸡卵清白蛋白（ovalbumin, OVA）及其多克隆抗体建立 RBL-2H3活化模型。结果显示， OVA多抗致敏再以OVA攻击时，细胞出现明显的脱颗粒，但OFE对该过程并无抑制作用；（2）碱性促分泌素可以通过MAS相关G蛋白偶联受体（MAS-related G protein coupled receptors, Mrgprs）直接触发肥大细胞活化，本研究利用其典型配体Compound 48/80建立IgE非依赖的肥大细胞活化体内体外模型。在体外模型上，OFE能浓度依赖地抑制Compound 48/80诱导的大鼠腹腔原代肥大细胞和LAD2细胞脱颗粒；在体内模型上，单次腹腔注射OFE对Compound 48/80诱导的小鼠低体温具有显著保护作用。由于胞质钙（Ca²⁺_[c]）动员是肥大细胞脱颗粒的关键事件，我们随后测定了OFE对Ca²⁺_[c]的影响。结果显示，OFE能够抑制Compound 48/80引起的LAD2细胞Ca²⁺_[c]升高，但不影响静息状态Ca²⁺_[c]，提示OFE可能通过影响Mrgprs信号通路中相关蛋白的活化来抑制Ca²⁺_[c]升高，从而发挥抗肥大细胞活化的作用。

       综上，本研究首次较全面地探究了OFE抗巨噬细胞及肥大细胞活化的作用，并对其潜在分子机制做了初步探索。在抗巨噬细胞活化方面，OFE能够通过抑制NF-κB和AP-1两条信号通路降低促炎因子的转录，从而减少巨噬细胞炎症介质的生成；在抗肥大细胞活化方面，OFE能够通过降低Mrgprs介导的Ca²⁺_[c]升高来抑制肥大细胞脱颗粒。本研究结果丰富了桂花在1型和2型炎症方面的药理学作用，为其在食品药品方面的应用和开发提供了科学的依据。

The inflammatory response represents an immunological defense mechanism by which immune cells are mobilized to clear deleterious stimuli. Based on the effector cells involved and the nature of stimuli, immune systems are categorized into 3 major kinds of immunity (Type 1, Type 2 and Type 3 immunity), with Types 1 and 2 predominating in occurrence. Type 1 immunity provides an effective response against intracellular pathogens, including bacteria and viruses. Type 2 immunity is mobilized by the host to combat parasitic worms or deleterious environmental stimuli. Both Type 1 and Type 2 immunity initiate unique inflammatory responses, reflective of their distinct functional roles. Hence, Type 1 immunity is commonly synonymous with Type 1 inflammation, while Type 2 immunity correlates with Type 2 inflammation. The inflammatory response is an inevitable reaction of the host to eliminate harmful stimuli. However, uncontrolled intensity or duration of the inflammation leads to continuous activation of the immune cells thereby causing damage to the body (such as cytokine storm, atopic disease), and in severe cases, threatening the life (such as septicemia, anaphylactic shock). Thus, the research on exploring anti-inflammatory agents targeting Type 1 or Type 2 inflammation are not only clinically imperative but also a focal point in novel drug research.

O. fragrans flower is the dried flower of Osmanthus fragrans (Thunb.) Lour, which belongs to the family Oleaceae and commonly known as sweet osmanthus. It is a typical dual-purpose medicinal and edible flower, historically incorporated into various food and beverage. Among these, the wine of O. fragrans flower has gained great popularity due to its simple preparation and notable efficacy. Traditionally, O. fragrans flower is believed to possess properties of resolving phlegm, relieving pain, reducing swelling, rendering it useful in treating inflammatory conditions such as asthma, toothache, gastralgia, and dysentery. Modern research has identified that the flower is rich in flavonoids, polyphenols, and phenylethanoid glycosides, exhibiting pronounced antioxidant, anti-tumor, analgesic, and anti-diabetic effects with low toxicity. In terms of anti-inflammatory activity, studies have demonstrated that the extract of the flower could alleviate colitis in mice and various atopic diseases (including allergic airway inflammation, allergic rhinitis, and eczema), suggesting its potential role in combating Type 1 and/or Type 2 inflammation. Therefore, the present study explored the anti-inflammatory effects and mechanisms of edible O. fragrans flower on macrophages (the main effector cells of Type 1 inflammation) and mast cells (the main effector cells of Type 2 inflammation) , respectively.

The ethanol extract of O. fragrans flower (OFE) was firstly prepared. Next, we investigated the effect and mechanisms of OFE on lipopolysaccharide (LPS)-induced macrophage inflammatory responses. In endotoxemia model, a single-dose intraperitoneal administration of OFE significantly reduced serum levels of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). In LPS-stimulated mice primary peritoneal macrophages and RAW264.7 cell, OFE concentration-dependently reduced the production of pro-inflammatory mediators, including nitric oxide (NO), IL-6, MCP-1, and interleukin-1β (IL-1β). Mechanistic investigations revealed that OFE did not affect the activity of inducible nitric oxide synthase (iNOS), but rather reduced NO production by inhibiting the transcription and translation of the enzyme. OFE also significantly inhibited the transcription of IL-6, MCP-1, and IL-1β. Since the transcription of these pro-inflammatory mediators is regulated by nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), the study subsequently explored the effect of OFE on the activation of these two transcription factors. The results demonstrated that OFE exerted significant inhibitory effects on the activation of NF-κB and AP-1: OFE reduced the phosphorylation level of NF-κB inhibitory protein IκBα and prevented its degradation, thereby inhibiting NF-κB nuclear translocation and ultimately suppressing NF-κB signaling. Additionally, OFE inhibited AP-1 activation by reducing the phosphorylation levels of ERK, p38, and JNK which belong to the mitogen-activated protein kinases (MAPKs) family and responsible for regulating the activation of AP-1.

Moreover, the study preliminarily investigated the effects and mechanisms of OFE on IgE-dependent and IgE-independent mast cell activation. Specifically, IgE-dependent mast cell activation was initiated upon the crosslinking of IgE-FcεRI complexes with specific antigens. Results indicated that in anti-OVA serum sensitized-RBL-2H3 cells, supernatant β-hexosaminidase was significantly elevated after OVA challenge. However, OFE showed no inhibitory effect during this process. Besides, a number of alkaline secretagogues can directly trigger mast cell activation via MAS-related G protein coupled receptors (Mrgprs) independently of IgE. The in vitro and in vivo model of mast cell activation was established using Compound 48/80, the typical ligand of Mrgprs. Results demonstrated that OFE not only concentration-dependently inhibited the degranulation of rat peritoneal mast cells and LAD2 cells induced by Compound 48/80 but also exerted a significant protective effect against Compound 48/80-induced hypothermia in mice by a single intraperitoneal injection. As cytosolic Ca²⁺ (Ca²⁺_[c]) mobilization is a pivotal event in mast cell degranulation, we subsequently assessed the impact of OFE on Ca²⁺_[c]. Our findings revealed that OFE inhibited the elevation of Ca²⁺_[c] induced by Compound 48/80 in LAD2 cells without affecting the resting Ca²⁺_[c] levels, suggesting that OFE might exert its inhibitory effect on mast cell inflammatory responses by modulating the activation of relevant proteins in Mrgprs signaling pathway.

In summary, the present study provided a comprehensive investigation into the anti-inflammatory effects of OFE, particularly focusing on its effect of macrophage and mast cell activation, along with a preliminary exploration of its underlying molecular mechanisms. Regarding the inhibition of macrophage activation, OFE demonstrated the capacity to reduce the transcription of pro-inflammatory cytokines by suppressing the NF-κB and AP-1 signaling pathways, consequently suppressing the production of these mediators. Concerning the suppression of mast cell activation, OFE exhibited its inhibitory effect on mast cell degranulation by downregulating the Mrgprs-mediated elevation of Ca²⁺_[c]. These findings enriched the pharmacological understanding of the flower of O. fragrans in Type 1 and Type 2 inflammation, thus providing a scientific basis for its potential applications and development in the food and pharmaceutical industries.