一种新的萘酚类衍生物1-羟基-2-萘甲酸甲酯的抗炎作用及其作用机制的研究
|
摘要
炎症是机体组织对损伤性刺激的一种防御性反应,它是一个由多细胞(比如淋巴细胞、巨噬细胞、粒细胞、内皮细胞等)、多成分(细胞因子,血管活性物质、趋化因子、粘附因子、炎症相关酶类)参与的复杂的过程。在炎症过程中,一方面损伤因子直接或间接造成组织和细胞的破坏,另一方面通过炎症充血和渗出反应,以稀释、杀伤和包围损伤因子。同时通过实质和间质细胞的再生使受损的组织得以修复和愈合。因此炎症是损伤和抗损伤的统一过程。适当的炎症反应对人体抵抗损伤、清除感染,促进伤口的愈合是有益的,然而过度的炎症反应会导致持久性的组织损伤,进而影响我们的身体健康。
巨噬细胞是由血液中单核细胞迁入组织后分化而成的,它在不同器官、组织中均有分布,例如：肺中的肺泡巨噬细胞、肝脏中的枯否氏细胞、神经组织中的神经胶质细胞以及骨组织中的蚀骨细胞等等。巨噬细胞是机体内重要的一种免疫细胞,具有抗感染、抗肿瘤和免疫调节的作用。同时它又是体内启动炎症介质产生的中心细胞。巨噬细胞能够被多种炎症性刺激所激活,其中就包括作用较强的细菌脂多糖。细菌脂多糖又称内毒素,是革兰氏阴性细菌致病的主要因素,具有较强激活单核巨噬细胞的作用。当革兰氏阴性细菌在死亡或进行繁殖时释放出来的LPS进人宿主体内,可诱导单核巨噬细胞合成并释放多种炎症介质,诸如：NO、前列腺素PGs以及促炎性细胞因子TNF-α、IL-1β、IL-6等,导致机体出现一系列的炎症反应,严重的可以出现中毒性休克,全身炎症反应综合征以及多器官功能衰竭等。巨噬细胞及其释放的炎症介质在炎症过程中扮演了一个重要的角色。目前研究认为巨噬细胞参与了多种炎症性疾病的发生与发展,例如,动脉粥样硬化、风湿性关节炎、炎症性肠病、败血症等。
NO是一种毒性较强的气体自由基,其在体内由一氧化氮合酶(nitric oxide synthase, NOS)催化左旋精氨酸而产生,具有广泛的生物学功能。NO是重要的细胞信号分子,同时还参与神经信息传递、心肺功能调节、细胞凋亡以及免疫防御等多个过程。目前己知的NOS有神经型NOS(nNOS)、内皮型NOS(eNOS)、诱生型NOS(iNOS),前两者又称为结构型NOS(cNOS),由cNOS催化生成的NO浓度很低,主要参与生理过程,维持细胞的正常功能。而巨噬细胞产生的NO浓度高,主要由诱导性一氧化氮合酶催化产生。短期、大量的NO释放使巨噬细胞具有杀伤微生物以及肿瘤细胞的作用,然而长期、过度的NO会通过DNA损伤、线粒体呼吸抑制、活性氮等细胞毒性效应介导细胞和组织的损伤,进而参与某些炎症性疾病的发生与发展。因此,抑制NO的过量表达已经成为预防炎症反应和疾病的研究热点。
前列腺素(prostaglandins, PGS)是广泛存在于动物和人体内的一组重要的组织激素,不仅对心血管、胃肠道、呼吸和生殖系统的功能有强烈的调节作用,而且还是炎症、发热及疼痛发生和发展的一个关键介质。环氧化酶(cyclooxygenase, COX)是催化花生四烯酸生成前列腺素的关键酶,包括COX-1、 COX-2和COX-3三种亚型。COX-1和COX-3为结构型环氧化酶,主要存在于血管、胃、肾等组织中,参与血管舒缩、血小板聚集、胃粘膜血流、胃黏液分泌及肾功能等的调节,其功能与保护胃肠黏膜、调节血小板聚集、调节外周血管的阻力和调节肾血流量分布有关。COX-2为诱导型环氧化酶,在正常组织中几乎不表达,但在活化的巨噬细胞中却诱导性地大量表达。传统的非淄体抗炎药例如阿司匹林、罗非昔布等通过抑制环氧化酶的活性,减少了前列腺素的产生,进而具有解热镇痛抗炎的作用。这一类药物已经被广泛的应用于治疗风湿性关节炎以及其他炎症性疾病当中。
TNF-α、IL-1β以及IL-6均是促炎性细胞因子,它们可以直接激活免疫效应细胞(如T细胞、B细胞、巨噬细胞、NK细胞等),也可以旁分泌或自分泌的形式促进其他炎症介质的产生,进而扩大炎症反应。正常水平的TNF-α、IL-1β以及IL-6对调节免疫应答、抗感染、促进组织修复、引起肿瘤细胞凋亡等至关重要,然而促炎性细胞因子的大量产生和释放则会破坏机体的免疫平衡,引起过度的炎症反应,介导炎症性疾病的发生。例如,某些炎症性疾病如类风湿关节炎患者血液循环当中的促炎性细胞因子就有不同程度的增加,其水平与疾病的严重程度往往具有一定的相关性。而且通过使用单克隆抗体或受体拮抗剂阻断促炎性细胞因子介导的效应,在治疗某些炎症性疾病上已取得了良好的治疗效果。
巨噬细胞产生上述炎症介质是通过复杂的细胞内信号转导通路的激活而实现的,其中与巨噬细胞炎症基因和蛋白表达关系最密切的是转录因子NF-κ B和有丝分裂原蛋白激酶MAPKs.
NF-κB (nuclear factor-κB)是一类广泛存在于哺乳动物细胞中、具有多向转录调节作用的核转录因子,它能够参与机体的炎症反应、免疫反应、细胞分化与凋亡及其它应激反应。在哺乳动物细胞中,NF-κB通常以p50/p65异源二聚体形式存在。当细胞在静息状态下,NF-κB与其抑制性蛋白Ⅰκ B结合,并以三聚体的无活性形式存在于胞浆中。当细胞受到外来刺激时,Ⅰκ B发生磷酸化并被降解,NF-κB被解除抑制从胞浆转移到细胞核中,并与靶基因上游调控序列结合,启动相关基因的转录。NF-κB的激活主要是通过Ⅰκ B的降解来调节的,然而越来越多的研究表明：NF-κB的活性调节还可以通过NF-κB蛋白的磷酸化和乙酞化等直接修饰作用来实现。例如酪蛋白激酶Ⅱ能够通过磷酸化p65亚基的529位丝氨酸残基,增强NF-κB的转录活性。
NF-κ B信号传导通路是LPS所介导的信号传导通路中最重要的下游通路。也是巨噬细胞激活的中心环节。当巨噬细胞NF-κ B被激活后,可迅速诱导炎症细胞因子、粘附分子、趋化分子以及炎症相关酶类的表达。如TNF-α、IL-1β、 IL-6、iNOS、COX-2等。因此阻断NF-κB信号通路的活化,可以减少多种炎症介质的产生,从而发挥抗炎的作用。
丝裂原活化蛋白激酶(mitogen activated protein kinases, MAPKs)是细胞内的一类丝氨酸和(或)苏氨酸蛋白激酶,是细胞应激和损伤反应的主要信号通路,在炎症信号转导、细胞凋亡、肿瘤细胞增殖及参与调控体内多种物质代谢过程中起着重要作用。MAPKs家族有三条经典的亚族通路,分别是细胞外调节蛋白激酶1/2(extracellular Signal-regulated kinasesl/2,ERKI/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)、p38丝裂原活化蛋白激酶(p38Mitogen-Activated Protein Kinases, p38-MApK)。MAPKs的激活(主要是通过磷酸化)是LPS作用于单核/巨噬细胞后发生的细胞内早期事件之一,而且MAPKs的底物多为核转录因子,其中就包括NF-κ B、AP-1等,因此MAPKs与炎性细胞因子的合成和分泌密切相关。
巨噬细胞激活及其释放的炎症介质在炎症性疾病当中发挥了重要的作用,因此抑制活化巨噬细胞产生炎症介质已经成为抗炎药物治疗和研究的靶点。而且更让人关注的是：以抑制巨噬细胞激活和抑制巨噬细胞过度释放炎症介质为靶点的这类抗炎药物,与传统的抗炎药物相比在理论有自己独特的优势。
在我国,抗炎药物是仅次于抗感染药物的第二大类药物。目前的抗炎免疫药物主要分为三大类,即甾体类抗炎药和非甾体类抗炎药(NSAIDs)以及疾病调修药,三类抗炎免疫药物在临床上已经被广泛地应用。甾体类抗炎药主要有糖皮质激素类药物如地塞米松等,具有广泛的生理和药理作用,但长期应用有引起物质代谢和水盐代谢紊乱,诱发或加重感染,导致骨质疏松等毒副作用；非甾体类抗炎药物主要是通过抑制COX进而抑制前列腺素(PGs)的产生,而发挥抗炎作用。可细分为非选择性COX抑制剂(如阿司匹林)以及选择性COX-2抑制剂(如塞来昔布)等,非选择性COX抑制剂因为选择性低,所以会引起很多的毒副反应,选择性COX-2抑制剂毒副作用少,然而随着研究发现COX-2在大脑、精囊及肾脏中也存在结构型表达,如果抑制这些组织中的COX-2活性会产生哪些负面的影响,需要引起大家进一步的重视。疾病调修药包括免疫抑制剂、免疫增强剂以及免疫调节剂,这类药物对免疫功能有抑制或增强或双向调节的作用。例如、雷帕霉素以及来氟米特等具有抑制免疫反应的作用,主要用于器官移植的抗排斥反应；左旋咪唑作为免疫促进剂主要用于自身免疫性疾病的治疗,还可作为肿瘤治疗的辅助用药等。
随着抗炎免疫药理学研究的不断深入以及人们在炎症反应细胞分子机制方面取得的研究进展,迫切需要我们寻找更多抗炎效果好、毒副作用少,能够长期应用的抗炎药物。然而抑制巨噬细胞激活及其介导的炎症反应就是一个非常好的以细胞为整体的药物研究靶点。
萘酚类衍生物是一类在萘环上含有羟基的化合物,它们作为原料药在医药与工业行业当中被广泛地应用。整理文献发现：萘酚类衍生物的研究文献主要集中在化学层面、而有关其活性方面的文章并不多。现有已经报道的萘酚类衍生物活性主要体现在以下几个方面：抗细菌和真菌的活性；干扰糖的无氧酵解；促进胆汁分泌、抗肿瘤活性以及抗炎活性。鉴于萘酚类衍生物结构与功能的多样性,这类化合物的抗炎活性尤为让人关注。研究发现2-取代-1-萘酚类衍生物具有抑制环氧化酶和脂氧合酶的作用。1,4-二羟基萘甲酸具有抑制骨吸收以及DSS诱导结肠炎的作用。
基于上述研究背景,我们准备以脂多糖诱导的巨噬细胞炎症模型以及酵母多糖诱导的小鼠腹膜炎模型,对萘酚类衍生物1-羟基2-萘甲酸甲酯(Methyl-1-hydroxy-2-naphthoate,MHNA)的体内外的抗炎活性及其可能的作用机制进行了深入的探讨。
主要研究方法和结果分为三个部分：
一、MHNA抑制脂多糖诱导的巨噬细胞炎症反应
1、MHNA对巨噬细胞细胞活力没有明显影响。
采用MTT法首先分析0-40μg/ml浓度范围内的MHNA对RAW264.7细胞活力的影响。MHNA在剂量达到40μg/ml浓度与巨噬细胞RAW264.7作用24h时,对巨噬细胞活力没有明显影响(差异无统计学意义)。因此本研究选取的MHNA最大剂量10μg/ml对巨噬细胞是安全的。
2、MHNA抑制LPS激活的巨噬细胞产生NO、IL-1β以及IL-6。
以2.5、5.0、10.0μg/ml MHNA以及0.1μ g/ml的LPS刺激RAW264.7细胞24h。留取细胞上清。用Griess试剂法检测细胞培养基中的NO, ELISA方法检测细胞上清中炎症细胞因子TNF-a, IL-1β以及IL-6飞水平。基础水平的NO浓度在2.69±0.39μM.,100μ g/ml的LPS作用24h巨噬细胞产生NO的浓度达到了21.78±1.24μM. MHNA在2.5-10μg/ml浓度范围内明显抑制LPS诱导的巨噬细胞产生NO。而且MHNA剂量依赖性地抑制LPS诱导地IL-1p以及IL-6的产生。然而它对巨噬细胞产生的TNF-a的影响相对较小,只在高浓度10μg/ml才有轻微地抑制作用,且统计学无显著性差异。
3、MHNA抑制LPS诱导的巨噬细胞iNOS及COX-2蛋白的表达
以2.5.5.0.10.0μg/ml MHNA以及0.1μg/ml的LPS刺激RAW264.7细胞24h。提取细胞总蛋白,用western blot方法探讨MHNA对巨噬细胞激活后诱导性酶iNOS及COX-2表达的影响。结果表明,MHNA显著抑制LPS诱导的巨噬细胞iNOS及COX-2蛋白的表达。
4. MHNA抑制LPS诱导的巨噬细胞炎症基因的表达
以2.5、5.0.10.0μg/ml MHNA以及终浓度为0.1μg/ml的LPS刺激RAW264.7细胞6h,提取细胞总RNA,经逆转录后,采用realtime-PCR方法,检测细胞炎症基因iNOS, COX-2, IL-1β以及IL-6mRNA的表达。结果表明,MHNA对lps诱导的巨噬细胞炎症基因iNOS, COX-2, IL-1β以及IL-6mRNA的表达有明显地抑制作用。
二、MHNA体内抗炎活性的研究
1. MHNA抑制二甲苯诱导的小鼠耳廓肿胀
为了探讨MHNA对急性炎症渗出的影响,我们构建了二甲苯诱导的小鼠耳廓肿胀模型。结果表明,MHNA20.40.80mg/kg的剂量灌胃给药,对二甲苯所致小鼠耳廓肿胀均有明显的抑制作用(P<0.05),且呈明显的量效关系,抑制率分别为17.8%、29.5%,45.0%。
2MHNA减轻腹膜炎小鼠腹腔中蛋白与白细胞的渗出。
为了进一步探讨MHNA的体内抗炎活性,我们构建了酵母多糖诱导的小鼠腹膜炎模型。研究表明：酵母多糖可以迅速诱导小鼠炎症反应,引起小鼠腹腔灌洗液中出现大量的蛋白与白细胞。地塞米松1Omg/kg以及MHNA40mg/kg, MHNA80mg/kg能显著性抑制腹腔中蛋白的渗出(抑制率分别是：45.9%,19.3%以及29.4%),而且还能抑制白细胞的浸润(抑制率分别是：57.4%,22.5%以及38.6%)。
3MHNA抑制腹膜炎小鼠腹腔中炎症细胞因子IL-6的水平
为了探讨MHNA对酵母多糖诱导的腹膜炎小鼠腹腔中炎症细胞因子产生的影响,我们检测了小鼠腹腔灌洗液中细胞因子IL-6的水平。研究结果表明：正常的小鼠腹腔中几乎检测不出IL-6,但是腹腔注射酵母多糖后6h,腹腔IL-6水平明显增高。地塞米松1Omg/kg以及MHNA40mg/kg, MHNA80mg/kg显著性抑制腹腔中IL-6的产生(抑制率分别是：48.7%,19.5%以及41.2%)。
三、MHNA抗炎作用机制的探讨
1、MHNA抑制巨噬细胞NF-κB的激活
为了探讨MHNA抑制巨噬细胞炎症反应的作用机制,我们分别用western blot方法,报告基因方法,以及凝胶迁移实验探讨了MHNA对巨噬细胞转录调节因子NF-κB激活的影响。RAW264.7细胞先与2.5、5.0、10.0μg/ml MHNA作用1h,然后再与0.1μg/ml的LPS作用30min(免疫印迹法检测iKB降解以及MAPKs的磷酸化)或1h(提取胞浆胞核蛋白,免疫印迹法检测胞浆胞核中NF-κ B的分布,凝胶迁移实验检测NF-κ B与DNA的结合能力)
Western blot结果显示：与未刺激的对照细胞相比,脂多糖刺激巨噬细胞后,细胞内iκB降解明显,胞浆内NF-κB减少,胞核内NF-κB增多,表明脂多糖刺激可以诱导NF-κB由胞浆向胞核内转移。MHNA与脂多糖一起作用,明显减弱了脂多糖引起的iκB降解、胞浆内NF-κB减少以及胞核内NF-κB增多的这一作用。
报告基因结果显示：0.1μg/ml脂多糖引起巨噬细胞NF-κB转录活性(反映为报告基因最后检测的相对荧光信号的强弱)相比对照细胞提高了5.3倍,而MHNA对巨噬细胞的转录活性有明显地抑制。
凝胶迁移实验EMSA结果显示：脂多糖引起巨噬细胞NF-κ B与其特异性结合的DNA序列结合能力增加,MHNA或NF-κB的特异性抑制剂BAY11-7082预处理巨噬细胞1h,均可以抑制脂多糖引起的NF-κB与DNA结合能力的增强。
2、MHNA抑制巨噬细胞MAPKs的激活
RAW264.7细胞先与2.5、5.0、10.0μg/ml MHNA作用1h,然后再与0.1μg/ml的LPS作用30min,提取细胞总蛋白,免疫印迹法检测MAPKs (p38MAPK, ERK1/2,以及JNK)的磷酸化。结果表明：脂多糖可以引起巨噬细胞p38MAPK, ERK1/2,以及JNK的磷酸化增加,而对总的p38MAPK, ERK1/2,以及JNK的蛋白水平没有明显影响。2.5-10.0μg/ml的MHNA剂量依赖性地抑制p38MAPK以及JNK的磷酸化,而对ERK1/2的磷酸化水平没有明显影响。
结论：
一、本文首次发现,MHNA具有抑制LPS诱导的巨噬细胞炎症反应的作用。具体表现为：
1、MHNA抑制激活的巨噬细胞炎症介质NO、IL-1β以及IL-6的产生；
2、MHNA抑制激活的巨噬细胞iNOS与COX-2蛋白的表达；
3、MHNA抑制激活的巨噬细胞炎症基因(iNOS、COX-2、IL-1β、IL-6)的表达。
二、MHNA具有良好的体内抗炎活性。
具体表现为：
1、MHNA抑制了二甲苯诱导的小鼠耳廓肿胀
2、MHNA减轻了酵母多糖诱导的腹膜炎小鼠腹腔中蛋白与白细胞的渗出。
3、MHNA抑制了酵母多糖诱导的腹膜炎小鼠腹腔中炎症细胞因子IL-6的水平。
三、MHNA通过抑制巨噬细胞NF-κB、p38MAPK以及JNK信号通路的激活进而抑制巨噬细胞激活及其介导的炎症反应。
综上所述：MHNA通过阻断NF-κ B、p38MAPK以及JNK信号通路的活化,抑制了巨噬细胞的激活,减少了巨噬细胞炎症介质的产生,从而对巨噬细胞介导的炎症反应有良好的抑制作用。而且其抗炎活性在体内炎症模型：二甲苯诱导的小鼠耳廓肿胀以及酵母多糖诱导的小鼠腹膜炎中得到了更进一步的体现。本研究为探讨萘酚类衍生物的体内外抗炎活性及其作用机制提出了新的实验依据。
Inflammation is a defensive reaction of the body tissues against to injury stimulus; it is a complicated process that involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation is a unified process of injury and damage resistance. Appropriate inflammatory response is helpful for the human body to resist damage, clear the infection and promote wound healing; however, the excessive inflammatory response will lead to persistent tissue damage, thereby affecting our health.
Macrophages are differentiated from blood monocytes after monocytes moving into the tissues. They are widely distributed in different organs, for example:alveolar macrophages in lungs, Kupffer cells in liver, glial cells in nervous system and osteoclast in bone tissue. Macrophages are important immune cells in the body; they have anti-infection, anti-tumor and immunomodulatory effects. But they are also the center cell to produce inflammatory mediators. Macrophages can be activated by various inflammatory stimuli; including bacterial lipopolysaccharide (also known as endotoxin).which is a pathogenic factor of gram-negative bacteria. When LPS released from the death or reproduction gram-negative bacteria which had been entered into the host body, it can induce monocyte-macrophage synthesis and to release various inflammatory mediators, such as NO, prostaglandins, and pro-inflammatory cytokine TNF-alpha, IL-1β, and IL-6etc., causing the body to a series of inflammatory reactions, which seriously can lead to severe toxic shock, systemic inflammatory response syndrome and multiple organ failure. Macrophage and its secreted inflammatory mediators play important roles in inflammatory process. The current studies suggested that macrophages were involved in the occurrence and development of a variety of inflammatory diseases such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, sepsis, and so on.
NO is a toxic gas free radicals, generated from L-arginine catalyzed by nitric oxide synthase (NOS).it possesses a wide range of biological functions. For example, NO is involved in processes of neural signaling transmission, function regulation of heart and lung, apoptosis and immune defense. Currently known of NOS include nerves nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible nitric oxide synthase (iNOS). The first two are also known as the constitutively nitric oxide synthase (cNOS). Low concentrations of NO generated by cNOS catalytic mainly involved in physiological processes; maintain the normal function of cells. High concentrations of NO produced by macrophages are mainly catalyzed by inducible nitric oxide synthase. Short-term, large amount of NO released by macrophage are benefit for killing microbes and tumor cells, however, long-term, excessive NO produced by macrophage mediates organizations injury through DNA damage, inhibition of mitochondrial respiration, reactive nitrogen and other cytotoxic effect, participate the occurrence and development of certain inflammatory diseases. Therefore, inhibition of NO over-production has became a hotspot for the prevention of inflammatory diseases.
As a group of important tissue hormones, prostaglandins (PGS) are widely present in animals and humans.They are not only play a strong role in regulating the function of the cardiovascular, gastrointestinal, respiratory and reproductive systems, but also critical in development of fever and pain in inflammation. Cyclooxygenases (COX)(including COX-1, COX-2and COX-3subtypes) are key enzyme catalyzing arachidonic acid to generate prostaglandins. Structure cyclooxygenase COX-1and COX-3are mainly present in blood vessels, stomach, kidney and other tissues. They are involved in vasomotion, platelet aggregation, and regulation of gastric mucosal blood flow, gastric mucus secretion and renal function. The inducible cyclooxygenase COX-2is low expression in normal tissues but large expression in activated macrophages. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, rofecoxib act as antipyretic analgesic and anti-inflammatory drug by inhibiting the activity of cyclooxygenase and reducing the production of prostaglandins. They have been widely used in the treatment of rheumatoid arthritis and other inflammatory diseases.
TNF-alpha, IL-1β and IL-6are proinflammatory cytokines, which can directly activate immune effector cells (such as T cells, B cells, macrophages, NK cells, etc.). Furthermore, they also promote the production of other inflammatory mediators in a paracrine or autocrine system, and thus amplify the inflammatory response. Normal levels of TNF-alpha, IL-1β, and IL-6is essential to modulate the immune response, anti-infection, promote tissue repair, and cause tumor cell apoptosis. However, excessive production and release of these pro-inflammatory cytokines will destroy the immune balance and cause excessive inflammatory response. For example, certain proinflammatory cytokines have been found increasing in the blood circulation of patients with inflammatory diseases such as rheumatoid arthritis, and the increased amount of these proinflammatory cytokines are correlation with the severity of diseases. Blocking the proinflammatory cytokine-mediated effects by using monoclonal antibodies or receptor antagonists has achieved good results in the treatment of many inflammatory diseases.
Macrophages produce the above-mentioned inflammatory mediators via activation of several complex intracellular signal transduction pathways. One of the most important is transcription factors NF-κB (nuclear factor-kappa B) signaling pathway. NF-κB is widely present in mammalian cells, and it participates in inflammation response, immune response as well as cell differentiation and apoptosis. In mammalian cells, NF-κB is usually existed as p50/p65heterodimer. In resting state, NF-κB exists as an inactive form in the cytoplasm by binding to its inhibitory kappa B (IκB) protein. Once cells are activated by external stimuli, IκB protein is degraded; NF-κB rapidly translocates into the nucleus, binds to the promoter elements of its targeted genes, and then regulates transcription of various genes. NF-kB signaling pathway activation is mainly regulated by the degradation of IκB; however, numerous studies have shown that NF-kB activity can also be regulated by direct modification of NF-kB protein by phosphorylation and acetylation. For example, casein kinase Ⅱ can enhance the transcriptional activity of NF-kB through phosphorylation of serine residue of p65subunit.
NF-κB signaling pathway is the most important downstream in the LPS-mediated signal transduction pathway. When the NF-κB signaling pathway in macrophage is activated, the expression of inflammatory cytokines, adhesion molecules, chemoattractant molecules and inflammation-related enzymes can be rapidly induced. Therefore, blocking the activation of NF-κB signaling pathway can reduce the production of various inflammatory mediators.
The mitogen activated protein kinases (MAPKs) are cellular serine and (or) threonine protein kinases. They play important roles in cell stress, damage responses, inflammatory signal transduction, cell apoptosis, tumor cell proliferation and material metabolism. The MAPKs family has three classic pathway of the subfamily, they are extracellular regulated protein kinase1/2(ERK1/2), c-Jun N-terminal kinase (JNK), p38mitogen-activated protein kinase (p38-MApK). The activation of MAPKs is one of the early events in the cells after LPS treatment in monocytes and macrophages. Furthmore, many important substrates for MAPKs are transcription factors such as NF-κB and AP-1.thus MAPKs are closely related to the synthesis and secretion of inflammatory mediators.
Excessive release of inflammatory mediators by activated macrophages is critical involved in inflammatory diseases. Therefore, inhibition of inflammatory mediators'production in activated macrophages has became a good target for anti-inflammatory drug research. Furthermore, drugs targeted at inhibition of macrophages activation and mediators release have their own unique advantages in theory compared with the traditional anti-inflammatory drugs.
In China, anti-inflammatory drugs are the second largest category of drugs followed anti-infective drugs in a certain peroid. The anti-inflammatory-immunity drugs are divided into three categories, steroidal anti-inflammatory drugs non-steroidal anti-inflammatory drugs (NSAIDs) and Disease-Modifying Drugs (DMDs). These three types of anti-inflammatory-immunity drugs have been widely applied in clinical practice. Steroidal anti-inflammatory drugs such as dexamethasone have a wide range of physiological and pharmacological effects, but long-term applications of steroidal anti-inflammatory drugs caused the disorder of metabolism, induced or exacerbated infection, lead to osteoporosis, etc. Non-steroidal anti-inflammatory drugs exert anti-inflammatory effect by inhibition of COX and thus inhibit the production of prostaglandins (PGs). They can be divided into non-selective COX inhibitors (such as aspirin) and selective COX-2inhibitor (such as celecoxib). COX inhibitors will cause a lot of side effects compared with the selective COX-2inhibitors. However, further studies found that COX-2is also structural expression in brain, seminal vesicle, and kidneys. The negative impacts by inhibition of COX-2activity in these tissues should pay more attention. Disease-Modifying Drugs contained immunosuppressants, immunostimulants and immunomodulatory agents, For example, rapamycin and leflunomide suppressed the immune response, which mainly be used as anti-rejection agent in organ transplantation; levamisole as an immunostimulants is used for the treatment of autoimmune diseases or as an adjuvant in cancer treatment.
With the continuous deepening of the anti-inflammatory immunopharmacological research, we are urgently to find more drugs with good anti-inflammatory activity and fewer side effects. Targeting at the inhibition of macrophage activation and its mediators release may be a good choice.
Naphthol derivatives are widely used in medical and industrial applications. They have a naphthol nucleus as a chemical backbone. Some naphthol derivatives have been reported to display a variety of biological activities. For instance,2-substituted-l-naphthol derivatives were shown to inhibit the activities of cyclooxygenase and5-lipooxygenase. A metabolic naphthol derivative,1,4-dihydroxy-2-naphthoic acid, from fermentation by Propionibacterium freudenreichii was reported to suppress bone resorption and dextran sodium sulphate (DSS)-induced colitis. Given the diversity of chemical structure and biological activity of naphthol derivatives, their anti-inflammatory activity is particularly attracted us. Thus, in this study, the LPS-induced macrophage inflammatory model and zymosan-induced mouse peritonitis model are carried out to explore the anti-inflammatory activity of a novel naphthol derivatives Methyl-1-hydroxy-2-naphthoate in vitro and in vivo, and its possible mechanism are also investigated.
The major research methods and results are divided into three parts:
part I:MHNA inhibited LPS-induced macrophage inflammatory response
1, MHNA had no significant effect on cell viability of macrophages.
MTT assay was firstly used to analyze the effect of MHNA (0-40μg/ml) on RAW264.7cell viability. MHNA at dose of40μg/ml cocultured with macrophage cell line RAW264.7for24h had no significant effect on macrophage viability (no significant difference in statistically). Thus the maximum dose10μg/ml of MHNA is safe for RAW264.7macrophages.
2, MHNA inhibited NO, IL-1β, and IL-6production in LPS-activated macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. NO and inflammatory cytokine TNF-a, IL-1β and IL-6in cell culture medium were detected by Griess reagent and ELISA methods, respectively. The basal level of NO, TNF-a, IL-1βp and IL-6in cell culture medium is very low. After macrophages being stimulated with100μg/ml LPS for24h, NO, TNF-a, IL-1β and IL-6were increased obviously. MHNA at dose of2.5-10μg/ml significantly dose-dependently inhibited LPS-induced macrophage cells to produce NO, IL-1β and IL-6. However, TNF-a produced by LPS-activated macrophages was not significantly affected by MHNA.
3, MHNA inhibited iNOS and COX-2protein expression in LPS-induced macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. Total protein was extracted, and then iNOS and COX-2protein expression were investigated by western blot. The results showed that, MHNA significantly inhibited LPS-induced macrophage iNOS and COX-2protein expression in a dose dependent manner.
4, MHNA inhibited inflammatory genes expression in LPS-activated macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using TPIzol reagent. Then, inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA level was quantitated by reverse transcription and realtime-PCR methods. The results show that MHNA inhibited macrophage inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA expression.
part II:anti-inflammatory activity of MHNA in vivo.
1MHNA inhibited xylene-induced mouse ear swelling
In order to explore the impact of MHNA in acute inflammatory exudation, a xylene-induced mouse ear swelling test was used. The results show that, MHNA at dose of20,40,80mg/kg orally administered, significantly inhibited mouse ear swelling caused by xylene in a dose dependent manner (P<0.05). The inhibition rates were17.8%,29.5%,45.0%, respectively.
2MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.
To further investigate the anti-inflammatory activity of the MHNA in vivo, we constructed zymosan induced murine peritonitis model. In this study, zymosan rapidly induced inflammatory response in mice, causing a lot of protein exudation and leukocytes infiltration in the mouse peritoneal. Dexamethasone10mg/kg and MHNA40mg/kg, MHNA80mg/kg significantly inhibited protein exudation in the abdominal cavity (inhibition rate:45.9%,19.3%and29.4%), they also inhibited leukocyte infiltration (The inhibition rates were:57.4%,22.5%and38.6%).
3MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis.
In order to investigate the impact of MHNA on inflammatory cytokines production in zymosan A-induced peritonitis in the abdominal cavity of mice, we examined the cytokine IL-6level in the mouse peritoneal lavage fluid. The results show that IL-6almost can not be detected in peritoneal of normal mouse; but6h after intraperitoneal injection of zymosan, the abdominal cavity of IL-6levels were significantly increased. Dexamethasone10mg/kg, MHNA40mg/kg and MHNA80mg/kg significantly inhibited IL-6production in the abdominal cavity (inhibition rate were48.7%,19.5%and41.2%, respectively).
Part III anti-inflammatory mechanism of MHNA
1, MHNA inhibited NF-κB signaling pathway activation in macrophages
In order to explore the mechanism of MHNA inhibiting macrophage inflammatory response, NF-κB signaling pathway activation was detected by western blot, reporter gene assay and electrophoretic mobility shift assay. Pretreated RAW264.7cells with2.5,5.0,10.0μg/ml MHNA for lh, then cells were cocultured with0.1μg/ml LPS for30min (detection iκB degradation and MAPKs phosphorylation by Western blot) or1h (extracted cytosolic and nuclear proteins, then detected NF-κB distribution in the cytoplasm and nucleus by Western blot and investigated NF-κB DNA binding ability by electrophoretic mobility shift assay.
Western blot results showed that in LPS-stimulated macrophages, iκB protein degradated significantly, and the cytoplasm of NF-κB obviously reduced while NF-κB within the nucleus increased by compared with unstimulated control cells. This indicated that LPS stimulation induced NF-κB translocated from cytoplasm into the nucleus. Pretreatment with MHNA for1h, significantly attenuated the LPS-induced iκB degradation and NF-κB translocation.
Reporter gene results showed that0.1μg/ml LPS caused transcriptional activity of macrophages increased5.3-fold compared to control cells while MHNA significantly inhibited the increasing transcriptional activity of LPS-stimulated macrophages
The results of electrophoretic mobility shift assay showed that LPS increase NF-κB binding activity to its specific DNA sequences in macrophages. Pretreatment macrophages with MHNA and NF-κB specific inhibitor BAY11-7082for1h inhibited the increase of LPS-induced NF-κB and DNA binding activity.
2, MHNA inhibited MAPKs signaling pathway activation in macrophages
RAW264.7cells were pretreated with2.5,5.0,10.0μg/ml MHNA for1h, then cells were cocultured with0.1μg/ml LPS for30min. Total protein was extracted, and MAPKs (p38MAPK, ERK1/2and JNK) phosphorylation were detected by Western blot. The results showed that LPS increased p38MAPK, ERK1/2and JNK phosphorylation in macrophages.2.5-10.Oμg/ml MHNA dose-dependently inhibited p38MAPK and JNK phosphorylation, but had no significant effects on ERK1/2phosphorylation.
Conclusion:
1, MHNA inhibited LPS-induced macrophage inflammatory response.
A, MHNA inhibited inflammatory mediators NO, IL-1β and IL-6production in LPS activated macrophages;
B, MHNA inhibited iNOS and COX-2protein expression in LPS activated macrophages;
C, MHNA inhibited inflammatory genes (iNOS, COX-2, IL-1β and IL-6) expression in LPS activated macrophages.
2, MHNA showed a good anti-inflammatory activity in vivo.
A, MHNA inhibited mouse ear swelling caused by xylene
B, MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.
C, MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis.
3, MHNA inhibited macrophage activation by inhibiting NF-kappa B, p38MAPK and JNK signaling pathway activation.
In summary, MHNA inhibited inflammatory mediators'production in LPS-activated macrophages by blocking NF-κB, p38MAPK and JNK signaling pathway activation. Further studies found that MHNA also showed good anti-inflammtroy activity in xylene-induced mouse ear swelling test and zymosan-induced mouse peritonitis. These studies provide a new experimental basis to investigate the anti-inflammatory activity and mechanism of naphthol derivatives in vitro and in vivo.
巨噬细胞是由血液中单核细胞迁入组织后分化而成的,它在不同器官、组织中均有分布,例如：肺中的肺泡巨噬细胞、肝脏中的枯否氏细胞、神经组织中的神经胶质细胞以及骨组织中的蚀骨细胞等等。巨噬细胞是机体内重要的一种免疫细胞,具有抗感染、抗肿瘤和免疫调节的作用。同时它又是体内启动炎症介质产生的中心细胞。巨噬细胞能够被多种炎症性刺激所激活,其中就包括作用较强的细菌脂多糖。细菌脂多糖又称内毒素,是革兰氏阴性细菌致病的主要因素,具有较强激活单核巨噬细胞的作用。当革兰氏阴性细菌在死亡或进行繁殖时释放出来的LPS进人宿主体内,可诱导单核巨噬细胞合成并释放多种炎症介质,诸如：NO、前列腺素PGs以及促炎性细胞因子TNF-α、IL-1β、IL-6等,导致机体出现一系列的炎症反应,严重的可以出现中毒性休克,全身炎症反应综合征以及多器官功能衰竭等。巨噬细胞及其释放的炎症介质在炎症过程中扮演了一个重要的角色。目前研究认为巨噬细胞参与了多种炎症性疾病的发生与发展,例如,动脉粥样硬化、风湿性关节炎、炎症性肠病、败血症等。
NO是一种毒性较强的气体自由基,其在体内由一氧化氮合酶(nitric oxide synthase, NOS)催化左旋精氨酸而产生,具有广泛的生物学功能。NO是重要的细胞信号分子,同时还参与神经信息传递、心肺功能调节、细胞凋亡以及免疫防御等多个过程。目前己知的NOS有神经型NOS(nNOS)、内皮型NOS(eNOS)、诱生型NOS(iNOS),前两者又称为结构型NOS(cNOS),由cNOS催化生成的NO浓度很低,主要参与生理过程,维持细胞的正常功能。而巨噬细胞产生的NO浓度高,主要由诱导性一氧化氮合酶催化产生。短期、大量的NO释放使巨噬细胞具有杀伤微生物以及肿瘤细胞的作用,然而长期、过度的NO会通过DNA损伤、线粒体呼吸抑制、活性氮等细胞毒性效应介导细胞和组织的损伤,进而参与某些炎症性疾病的发生与发展。因此,抑制NO的过量表达已经成为预防炎症反应和疾病的研究热点。
前列腺素(prostaglandins, PGS)是广泛存在于动物和人体内的一组重要的组织激素,不仅对心血管、胃肠道、呼吸和生殖系统的功能有强烈的调节作用,而且还是炎症、发热及疼痛发生和发展的一个关键介质。环氧化酶(cyclooxygenase, COX)是催化花生四烯酸生成前列腺素的关键酶,包括COX-1、 COX-2和COX-3三种亚型。COX-1和COX-3为结构型环氧化酶,主要存在于血管、胃、肾等组织中,参与血管舒缩、血小板聚集、胃粘膜血流、胃黏液分泌及肾功能等的调节,其功能与保护胃肠黏膜、调节血小板聚集、调节外周血管的阻力和调节肾血流量分布有关。COX-2为诱导型环氧化酶,在正常组织中几乎不表达,但在活化的巨噬细胞中却诱导性地大量表达。传统的非淄体抗炎药例如阿司匹林、罗非昔布等通过抑制环氧化酶的活性,减少了前列腺素的产生,进而具有解热镇痛抗炎的作用。这一类药物已经被广泛的应用于治疗风湿性关节炎以及其他炎症性疾病当中。
TNF-α、IL-1β以及IL-6均是促炎性细胞因子,它们可以直接激活免疫效应细胞(如T细胞、B细胞、巨噬细胞、NK细胞等),也可以旁分泌或自分泌的形式促进其他炎症介质的产生,进而扩大炎症反应。正常水平的TNF-α、IL-1β以及IL-6对调节免疫应答、抗感染、促进组织修复、引起肿瘤细胞凋亡等至关重要,然而促炎性细胞因子的大量产生和释放则会破坏机体的免疫平衡,引起过度的炎症反应,介导炎症性疾病的发生。例如,某些炎症性疾病如类风湿关节炎患者血液循环当中的促炎性细胞因子就有不同程度的增加,其水平与疾病的严重程度往往具有一定的相关性。而且通过使用单克隆抗体或受体拮抗剂阻断促炎性细胞因子介导的效应,在治疗某些炎症性疾病上已取得了良好的治疗效果。
巨噬细胞产生上述炎症介质是通过复杂的细胞内信号转导通路的激活而实现的,其中与巨噬细胞炎症基因和蛋白表达关系最密切的是转录因子NF-κ B和有丝分裂原蛋白激酶MAPKs.
NF-κB (nuclear factor-κB)是一类广泛存在于哺乳动物细胞中、具有多向转录调节作用的核转录因子,它能够参与机体的炎症反应、免疫反应、细胞分化与凋亡及其它应激反应。在哺乳动物细胞中,NF-κB通常以p50/p65异源二聚体形式存在。当细胞在静息状态下,NF-κB与其抑制性蛋白Ⅰκ B结合,并以三聚体的无活性形式存在于胞浆中。当细胞受到外来刺激时,Ⅰκ B发生磷酸化并被降解,NF-κB被解除抑制从胞浆转移到细胞核中,并与靶基因上游调控序列结合,启动相关基因的转录。NF-κB的激活主要是通过Ⅰκ B的降解来调节的,然而越来越多的研究表明：NF-κB的活性调节还可以通过NF-κB蛋白的磷酸化和乙酞化等直接修饰作用来实现。例如酪蛋白激酶Ⅱ能够通过磷酸化p65亚基的529位丝氨酸残基,增强NF-κB的转录活性。
NF-κ B信号传导通路是LPS所介导的信号传导通路中最重要的下游通路。也是巨噬细胞激活的中心环节。当巨噬细胞NF-κ B被激活后,可迅速诱导炎症细胞因子、粘附分子、趋化分子以及炎症相关酶类的表达。如TNF-α、IL-1β、 IL-6、iNOS、COX-2等。因此阻断NF-κB信号通路的活化,可以减少多种炎症介质的产生,从而发挥抗炎的作用。
丝裂原活化蛋白激酶(mitogen activated protein kinases, MAPKs)是细胞内的一类丝氨酸和(或)苏氨酸蛋白激酶,是细胞应激和损伤反应的主要信号通路,在炎症信号转导、细胞凋亡、肿瘤细胞增殖及参与调控体内多种物质代谢过程中起着重要作用。MAPKs家族有三条经典的亚族通路,分别是细胞外调节蛋白激酶1/2(extracellular Signal-regulated kinasesl/2,ERKI/2)、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)、p38丝裂原活化蛋白激酶(p38Mitogen-Activated Protein Kinases, p38-MApK)。MAPKs的激活(主要是通过磷酸化)是LPS作用于单核/巨噬细胞后发生的细胞内早期事件之一,而且MAPKs的底物多为核转录因子,其中就包括NF-κ B、AP-1等,因此MAPKs与炎性细胞因子的合成和分泌密切相关。
巨噬细胞激活及其释放的炎症介质在炎症性疾病当中发挥了重要的作用,因此抑制活化巨噬细胞产生炎症介质已经成为抗炎药物治疗和研究的靶点。而且更让人关注的是：以抑制巨噬细胞激活和抑制巨噬细胞过度释放炎症介质为靶点的这类抗炎药物,与传统的抗炎药物相比在理论有自己独特的优势。
在我国,抗炎药物是仅次于抗感染药物的第二大类药物。目前的抗炎免疫药物主要分为三大类,即甾体类抗炎药和非甾体类抗炎药(NSAIDs)以及疾病调修药,三类抗炎免疫药物在临床上已经被广泛地应用。甾体类抗炎药主要有糖皮质激素类药物如地塞米松等,具有广泛的生理和药理作用,但长期应用有引起物质代谢和水盐代谢紊乱,诱发或加重感染,导致骨质疏松等毒副作用；非甾体类抗炎药物主要是通过抑制COX进而抑制前列腺素(PGs)的产生,而发挥抗炎作用。可细分为非选择性COX抑制剂(如阿司匹林)以及选择性COX-2抑制剂(如塞来昔布)等,非选择性COX抑制剂因为选择性低,所以会引起很多的毒副反应,选择性COX-2抑制剂毒副作用少,然而随着研究发现COX-2在大脑、精囊及肾脏中也存在结构型表达,如果抑制这些组织中的COX-2活性会产生哪些负面的影响,需要引起大家进一步的重视。疾病调修药包括免疫抑制剂、免疫增强剂以及免疫调节剂,这类药物对免疫功能有抑制或增强或双向调节的作用。例如、雷帕霉素以及来氟米特等具有抑制免疫反应的作用,主要用于器官移植的抗排斥反应；左旋咪唑作为免疫促进剂主要用于自身免疫性疾病的治疗,还可作为肿瘤治疗的辅助用药等。
随着抗炎免疫药理学研究的不断深入以及人们在炎症反应细胞分子机制方面取得的研究进展,迫切需要我们寻找更多抗炎效果好、毒副作用少,能够长期应用的抗炎药物。然而抑制巨噬细胞激活及其介导的炎症反应就是一个非常好的以细胞为整体的药物研究靶点。
萘酚类衍生物是一类在萘环上含有羟基的化合物,它们作为原料药在医药与工业行业当中被广泛地应用。整理文献发现：萘酚类衍生物的研究文献主要集中在化学层面、而有关其活性方面的文章并不多。现有已经报道的萘酚类衍生物活性主要体现在以下几个方面：抗细菌和真菌的活性；干扰糖的无氧酵解；促进胆汁分泌、抗肿瘤活性以及抗炎活性。鉴于萘酚类衍生物结构与功能的多样性,这类化合物的抗炎活性尤为让人关注。研究发现2-取代-1-萘酚类衍生物具有抑制环氧化酶和脂氧合酶的作用。1,4-二羟基萘甲酸具有抑制骨吸收以及DSS诱导结肠炎的作用。
基于上述研究背景,我们准备以脂多糖诱导的巨噬细胞炎症模型以及酵母多糖诱导的小鼠腹膜炎模型,对萘酚类衍生物1-羟基2-萘甲酸甲酯(Methyl-1-hydroxy-2-naphthoate,MHNA)的体内外的抗炎活性及其可能的作用机制进行了深入的探讨。
主要研究方法和结果分为三个部分：
一、MHNA抑制脂多糖诱导的巨噬细胞炎症反应
1、MHNA对巨噬细胞细胞活力没有明显影响。
采用MTT法首先分析0-40μg/ml浓度范围内的MHNA对RAW264.7细胞活力的影响。MHNA在剂量达到40μg/ml浓度与巨噬细胞RAW264.7作用24h时,对巨噬细胞活力没有明显影响(差异无统计学意义)。因此本研究选取的MHNA最大剂量10μg/ml对巨噬细胞是安全的。
2、MHNA抑制LPS激活的巨噬细胞产生NO、IL-1β以及IL-6。
以2.5、5.0、10.0μg/ml MHNA以及0.1μ g/ml的LPS刺激RAW264.7细胞24h。留取细胞上清。用Griess试剂法检测细胞培养基中的NO, ELISA方法检测细胞上清中炎症细胞因子TNF-a, IL-1β以及IL-6飞水平。基础水平的NO浓度在2.69±0.39μM.,100μ g/ml的LPS作用24h巨噬细胞产生NO的浓度达到了21.78±1.24μM. MHNA在2.5-10μg/ml浓度范围内明显抑制LPS诱导的巨噬细胞产生NO。而且MHNA剂量依赖性地抑制LPS诱导地IL-1p以及IL-6的产生。然而它对巨噬细胞产生的TNF-a的影响相对较小,只在高浓度10μg/ml才有轻微地抑制作用,且统计学无显著性差异。
3、MHNA抑制LPS诱导的巨噬细胞iNOS及COX-2蛋白的表达
以2.5.5.0.10.0μg/ml MHNA以及0.1μg/ml的LPS刺激RAW264.7细胞24h。提取细胞总蛋白,用western blot方法探讨MHNA对巨噬细胞激活后诱导性酶iNOS及COX-2表达的影响。结果表明,MHNA显著抑制LPS诱导的巨噬细胞iNOS及COX-2蛋白的表达。
4. MHNA抑制LPS诱导的巨噬细胞炎症基因的表达
以2.5、5.0.10.0μg/ml MHNA以及终浓度为0.1μg/ml的LPS刺激RAW264.7细胞6h,提取细胞总RNA,经逆转录后,采用realtime-PCR方法,检测细胞炎症基因iNOS, COX-2, IL-1β以及IL-6mRNA的表达。结果表明,MHNA对lps诱导的巨噬细胞炎症基因iNOS, COX-2, IL-1β以及IL-6mRNA的表达有明显地抑制作用。
二、MHNA体内抗炎活性的研究
1. MHNA抑制二甲苯诱导的小鼠耳廓肿胀
为了探讨MHNA对急性炎症渗出的影响,我们构建了二甲苯诱导的小鼠耳廓肿胀模型。结果表明,MHNA20.40.80mg/kg的剂量灌胃给药,对二甲苯所致小鼠耳廓肿胀均有明显的抑制作用(P<0.05),且呈明显的量效关系,抑制率分别为17.8%、29.5%,45.0%。
2MHNA减轻腹膜炎小鼠腹腔中蛋白与白细胞的渗出。
为了进一步探讨MHNA的体内抗炎活性,我们构建了酵母多糖诱导的小鼠腹膜炎模型。研究表明：酵母多糖可以迅速诱导小鼠炎症反应,引起小鼠腹腔灌洗液中出现大量的蛋白与白细胞。地塞米松1Omg/kg以及MHNA40mg/kg, MHNA80mg/kg能显著性抑制腹腔中蛋白的渗出(抑制率分别是：45.9%,19.3%以及29.4%),而且还能抑制白细胞的浸润(抑制率分别是：57.4%,22.5%以及38.6%)。
3MHNA抑制腹膜炎小鼠腹腔中炎症细胞因子IL-6的水平
为了探讨MHNA对酵母多糖诱导的腹膜炎小鼠腹腔中炎症细胞因子产生的影响,我们检测了小鼠腹腔灌洗液中细胞因子IL-6的水平。研究结果表明：正常的小鼠腹腔中几乎检测不出IL-6,但是腹腔注射酵母多糖后6h,腹腔IL-6水平明显增高。地塞米松1Omg/kg以及MHNA40mg/kg, MHNA80mg/kg显著性抑制腹腔中IL-6的产生(抑制率分别是：48.7%,19.5%以及41.2%)。
三、MHNA抗炎作用机制的探讨
1、MHNA抑制巨噬细胞NF-κB的激活
为了探讨MHNA抑制巨噬细胞炎症反应的作用机制,我们分别用western blot方法,报告基因方法,以及凝胶迁移实验探讨了MHNA对巨噬细胞转录调节因子NF-κB激活的影响。RAW264.7细胞先与2.5、5.0、10.0μg/ml MHNA作用1h,然后再与0.1μg/ml的LPS作用30min(免疫印迹法检测iKB降解以及MAPKs的磷酸化)或1h(提取胞浆胞核蛋白,免疫印迹法检测胞浆胞核中NF-κ B的分布,凝胶迁移实验检测NF-κ B与DNA的结合能力)
Western blot结果显示：与未刺激的对照细胞相比,脂多糖刺激巨噬细胞后,细胞内iκB降解明显,胞浆内NF-κB减少,胞核内NF-κB增多,表明脂多糖刺激可以诱导NF-κB由胞浆向胞核内转移。MHNA与脂多糖一起作用,明显减弱了脂多糖引起的iκB降解、胞浆内NF-κB减少以及胞核内NF-κB增多的这一作用。
报告基因结果显示：0.1μg/ml脂多糖引起巨噬细胞NF-κB转录活性(反映为报告基因最后检测的相对荧光信号的强弱)相比对照细胞提高了5.3倍,而MHNA对巨噬细胞的转录活性有明显地抑制。
凝胶迁移实验EMSA结果显示：脂多糖引起巨噬细胞NF-κ B与其特异性结合的DNA序列结合能力增加,MHNA或NF-κB的特异性抑制剂BAY11-7082预处理巨噬细胞1h,均可以抑制脂多糖引起的NF-κB与DNA结合能力的增强。
2、MHNA抑制巨噬细胞MAPKs的激活
RAW264.7细胞先与2.5、5.0、10.0μg/ml MHNA作用1h,然后再与0.1μg/ml的LPS作用30min,提取细胞总蛋白,免疫印迹法检测MAPKs (p38MAPK, ERK1/2,以及JNK)的磷酸化。结果表明：脂多糖可以引起巨噬细胞p38MAPK, ERK1/2,以及JNK的磷酸化增加,而对总的p38MAPK, ERK1/2,以及JNK的蛋白水平没有明显影响。2.5-10.0μg/ml的MHNA剂量依赖性地抑制p38MAPK以及JNK的磷酸化,而对ERK1/2的磷酸化水平没有明显影响。
结论：
一、本文首次发现,MHNA具有抑制LPS诱导的巨噬细胞炎症反应的作用。具体表现为：
1、MHNA抑制激活的巨噬细胞炎症介质NO、IL-1β以及IL-6的产生；
2、MHNA抑制激活的巨噬细胞iNOS与COX-2蛋白的表达；
3、MHNA抑制激活的巨噬细胞炎症基因(iNOS、COX-2、IL-1β、IL-6)的表达。
二、MHNA具有良好的体内抗炎活性。
具体表现为：
1、MHNA抑制了二甲苯诱导的小鼠耳廓肿胀
2、MHNA减轻了酵母多糖诱导的腹膜炎小鼠腹腔中蛋白与白细胞的渗出。
3、MHNA抑制了酵母多糖诱导的腹膜炎小鼠腹腔中炎症细胞因子IL-6的水平。
三、MHNA通过抑制巨噬细胞NF-κB、p38MAPK以及JNK信号通路的激活进而抑制巨噬细胞激活及其介导的炎症反应。
综上所述：MHNA通过阻断NF-κ B、p38MAPK以及JNK信号通路的活化,抑制了巨噬细胞的激活,减少了巨噬细胞炎症介质的产生,从而对巨噬细胞介导的炎症反应有良好的抑制作用。而且其抗炎活性在体内炎症模型：二甲苯诱导的小鼠耳廓肿胀以及酵母多糖诱导的小鼠腹膜炎中得到了更进一步的体现。本研究为探讨萘酚类衍生物的体内外抗炎活性及其作用机制提出了新的实验依据。
Inflammation is a defensive reaction of the body tissues against to injury stimulus; it is a complicated process that involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation is a unified process of injury and damage resistance. Appropriate inflammatory response is helpful for the human body to resist damage, clear the infection and promote wound healing; however, the excessive inflammatory response will lead to persistent tissue damage, thereby affecting our health.
Macrophages are differentiated from blood monocytes after monocytes moving into the tissues. They are widely distributed in different organs, for example:alveolar macrophages in lungs, Kupffer cells in liver, glial cells in nervous system and osteoclast in bone tissue. Macrophages are important immune cells in the body; they have anti-infection, anti-tumor and immunomodulatory effects. But they are also the center cell to produce inflammatory mediators. Macrophages can be activated by various inflammatory stimuli; including bacterial lipopolysaccharide (also known as endotoxin).which is a pathogenic factor of gram-negative bacteria. When LPS released from the death or reproduction gram-negative bacteria which had been entered into the host body, it can induce monocyte-macrophage synthesis and to release various inflammatory mediators, such as NO, prostaglandins, and pro-inflammatory cytokine TNF-alpha, IL-1β, and IL-6etc., causing the body to a series of inflammatory reactions, which seriously can lead to severe toxic shock, systemic inflammatory response syndrome and multiple organ failure. Macrophage and its secreted inflammatory mediators play important roles in inflammatory process. The current studies suggested that macrophages were involved in the occurrence and development of a variety of inflammatory diseases such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, sepsis, and so on.
NO is a toxic gas free radicals, generated from L-arginine catalyzed by nitric oxide synthase (NOS).it possesses a wide range of biological functions. For example, NO is involved in processes of neural signaling transmission, function regulation of heart and lung, apoptosis and immune defense. Currently known of NOS include nerves nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible nitric oxide synthase (iNOS). The first two are also known as the constitutively nitric oxide synthase (cNOS). Low concentrations of NO generated by cNOS catalytic mainly involved in physiological processes; maintain the normal function of cells. High concentrations of NO produced by macrophages are mainly catalyzed by inducible nitric oxide synthase. Short-term, large amount of NO released by macrophage are benefit for killing microbes and tumor cells, however, long-term, excessive NO produced by macrophage mediates organizations injury through DNA damage, inhibition of mitochondrial respiration, reactive nitrogen and other cytotoxic effect, participate the occurrence and development of certain inflammatory diseases. Therefore, inhibition of NO over-production has became a hotspot for the prevention of inflammatory diseases.
As a group of important tissue hormones, prostaglandins (PGS) are widely present in animals and humans.They are not only play a strong role in regulating the function of the cardiovascular, gastrointestinal, respiratory and reproductive systems, but also critical in development of fever and pain in inflammation. Cyclooxygenases (COX)(including COX-1, COX-2and COX-3subtypes) are key enzyme catalyzing arachidonic acid to generate prostaglandins. Structure cyclooxygenase COX-1and COX-3are mainly present in blood vessels, stomach, kidney and other tissues. They are involved in vasomotion, platelet aggregation, and regulation of gastric mucosal blood flow, gastric mucus secretion and renal function. The inducible cyclooxygenase COX-2is low expression in normal tissues but large expression in activated macrophages. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, rofecoxib act as antipyretic analgesic and anti-inflammatory drug by inhibiting the activity of cyclooxygenase and reducing the production of prostaglandins. They have been widely used in the treatment of rheumatoid arthritis and other inflammatory diseases.
TNF-alpha, IL-1β and IL-6are proinflammatory cytokines, which can directly activate immune effector cells (such as T cells, B cells, macrophages, NK cells, etc.). Furthermore, they also promote the production of other inflammatory mediators in a paracrine or autocrine system, and thus amplify the inflammatory response. Normal levels of TNF-alpha, IL-1β, and IL-6is essential to modulate the immune response, anti-infection, promote tissue repair, and cause tumor cell apoptosis. However, excessive production and release of these pro-inflammatory cytokines will destroy the immune balance and cause excessive inflammatory response. For example, certain proinflammatory cytokines have been found increasing in the blood circulation of patients with inflammatory diseases such as rheumatoid arthritis, and the increased amount of these proinflammatory cytokines are correlation with the severity of diseases. Blocking the proinflammatory cytokine-mediated effects by using monoclonal antibodies or receptor antagonists has achieved good results in the treatment of many inflammatory diseases.
Macrophages produce the above-mentioned inflammatory mediators via activation of several complex intracellular signal transduction pathways. One of the most important is transcription factors NF-κB (nuclear factor-kappa B) signaling pathway. NF-κB is widely present in mammalian cells, and it participates in inflammation response, immune response as well as cell differentiation and apoptosis. In mammalian cells, NF-κB is usually existed as p50/p65heterodimer. In resting state, NF-κB exists as an inactive form in the cytoplasm by binding to its inhibitory kappa B (IκB) protein. Once cells are activated by external stimuli, IκB protein is degraded; NF-κB rapidly translocates into the nucleus, binds to the promoter elements of its targeted genes, and then regulates transcription of various genes. NF-kB signaling pathway activation is mainly regulated by the degradation of IκB; however, numerous studies have shown that NF-kB activity can also be regulated by direct modification of NF-kB protein by phosphorylation and acetylation. For example, casein kinase Ⅱ can enhance the transcriptional activity of NF-kB through phosphorylation of serine residue of p65subunit.
NF-κB signaling pathway is the most important downstream in the LPS-mediated signal transduction pathway. When the NF-κB signaling pathway in macrophage is activated, the expression of inflammatory cytokines, adhesion molecules, chemoattractant molecules and inflammation-related enzymes can be rapidly induced. Therefore, blocking the activation of NF-κB signaling pathway can reduce the production of various inflammatory mediators.
The mitogen activated protein kinases (MAPKs) are cellular serine and (or) threonine protein kinases. They play important roles in cell stress, damage responses, inflammatory signal transduction, cell apoptosis, tumor cell proliferation and material metabolism. The MAPKs family has three classic pathway of the subfamily, they are extracellular regulated protein kinase1/2(ERK1/2), c-Jun N-terminal kinase (JNK), p38mitogen-activated protein kinase (p38-MApK). The activation of MAPKs is one of the early events in the cells after LPS treatment in monocytes and macrophages. Furthmore, many important substrates for MAPKs are transcription factors such as NF-κB and AP-1.thus MAPKs are closely related to the synthesis and secretion of inflammatory mediators.
Excessive release of inflammatory mediators by activated macrophages is critical involved in inflammatory diseases. Therefore, inhibition of inflammatory mediators'production in activated macrophages has became a good target for anti-inflammatory drug research. Furthermore, drugs targeted at inhibition of macrophages activation and mediators release have their own unique advantages in theory compared with the traditional anti-inflammatory drugs.
In China, anti-inflammatory drugs are the second largest category of drugs followed anti-infective drugs in a certain peroid. The anti-inflammatory-immunity drugs are divided into three categories, steroidal anti-inflammatory drugs non-steroidal anti-inflammatory drugs (NSAIDs) and Disease-Modifying Drugs (DMDs). These three types of anti-inflammatory-immunity drugs have been widely applied in clinical practice. Steroidal anti-inflammatory drugs such as dexamethasone have a wide range of physiological and pharmacological effects, but long-term applications of steroidal anti-inflammatory drugs caused the disorder of metabolism, induced or exacerbated infection, lead to osteoporosis, etc. Non-steroidal anti-inflammatory drugs exert anti-inflammatory effect by inhibition of COX and thus inhibit the production of prostaglandins (PGs). They can be divided into non-selective COX inhibitors (such as aspirin) and selective COX-2inhibitor (such as celecoxib). COX inhibitors will cause a lot of side effects compared with the selective COX-2inhibitors. However, further studies found that COX-2is also structural expression in brain, seminal vesicle, and kidneys. The negative impacts by inhibition of COX-2activity in these tissues should pay more attention. Disease-Modifying Drugs contained immunosuppressants, immunostimulants and immunomodulatory agents, For example, rapamycin and leflunomide suppressed the immune response, which mainly be used as anti-rejection agent in organ transplantation; levamisole as an immunostimulants is used for the treatment of autoimmune diseases or as an adjuvant in cancer treatment.
With the continuous deepening of the anti-inflammatory immunopharmacological research, we are urgently to find more drugs with good anti-inflammatory activity and fewer side effects. Targeting at the inhibition of macrophage activation and its mediators release may be a good choice.
Naphthol derivatives are widely used in medical and industrial applications. They have a naphthol nucleus as a chemical backbone. Some naphthol derivatives have been reported to display a variety of biological activities. For instance,2-substituted-l-naphthol derivatives were shown to inhibit the activities of cyclooxygenase and5-lipooxygenase. A metabolic naphthol derivative,1,4-dihydroxy-2-naphthoic acid, from fermentation by Propionibacterium freudenreichii was reported to suppress bone resorption and dextran sodium sulphate (DSS)-induced colitis. Given the diversity of chemical structure and biological activity of naphthol derivatives, their anti-inflammatory activity is particularly attracted us. Thus, in this study, the LPS-induced macrophage inflammatory model and zymosan-induced mouse peritonitis model are carried out to explore the anti-inflammatory activity of a novel naphthol derivatives Methyl-1-hydroxy-2-naphthoate in vitro and in vivo, and its possible mechanism are also investigated.
The major research methods and results are divided into three parts:
part I:MHNA inhibited LPS-induced macrophage inflammatory response
1, MHNA had no significant effect on cell viability of macrophages.
MTT assay was firstly used to analyze the effect of MHNA (0-40μg/ml) on RAW264.7cell viability. MHNA at dose of40μg/ml cocultured with macrophage cell line RAW264.7for24h had no significant effect on macrophage viability (no significant difference in statistically). Thus the maximum dose10μg/ml of MHNA is safe for RAW264.7macrophages.
2, MHNA inhibited NO, IL-1β, and IL-6production in LPS-activated macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. NO and inflammatory cytokine TNF-a, IL-1β and IL-6in cell culture medium were detected by Griess reagent and ELISA methods, respectively. The basal level of NO, TNF-a, IL-1βp and IL-6in cell culture medium is very low. After macrophages being stimulated with100μg/ml LPS for24h, NO, TNF-a, IL-1β and IL-6were increased obviously. MHNA at dose of2.5-10μg/ml significantly dose-dependently inhibited LPS-induced macrophage cells to produce NO, IL-1β and IL-6. However, TNF-a produced by LPS-activated macrophages was not significantly affected by MHNA.
3, MHNA inhibited iNOS and COX-2protein expression in LPS-induced macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. Total protein was extracted, and then iNOS and COX-2protein expression were investigated by western blot. The results showed that, MHNA significantly inhibited LPS-induced macrophage iNOS and COX-2protein expression in a dose dependent manner.
4, MHNA inhibited inflammatory genes expression in LPS-activated macrophages
RAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using TPIzol reagent. Then, inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA level was quantitated by reverse transcription and realtime-PCR methods. The results show that MHNA inhibited macrophage inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA expression.
part II:anti-inflammatory activity of MHNA in vivo.
1MHNA inhibited xylene-induced mouse ear swelling
In order to explore the impact of MHNA in acute inflammatory exudation, a xylene-induced mouse ear swelling test was used. The results show that, MHNA at dose of20,40,80mg/kg orally administered, significantly inhibited mouse ear swelling caused by xylene in a dose dependent manner (P<0.05). The inhibition rates were17.8%,29.5%,45.0%, respectively.
2MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.
To further investigate the anti-inflammatory activity of the MHNA in vivo, we constructed zymosan induced murine peritonitis model. In this study, zymosan rapidly induced inflammatory response in mice, causing a lot of protein exudation and leukocytes infiltration in the mouse peritoneal. Dexamethasone10mg/kg and MHNA40mg/kg, MHNA80mg/kg significantly inhibited protein exudation in the abdominal cavity (inhibition rate:45.9%,19.3%and29.4%), they also inhibited leukocyte infiltration (The inhibition rates were:57.4%,22.5%and38.6%).
3MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis.
In order to investigate the impact of MHNA on inflammatory cytokines production in zymosan A-induced peritonitis in the abdominal cavity of mice, we examined the cytokine IL-6level in the mouse peritoneal lavage fluid. The results show that IL-6almost can not be detected in peritoneal of normal mouse; but6h after intraperitoneal injection of zymosan, the abdominal cavity of IL-6levels were significantly increased. Dexamethasone10mg/kg, MHNA40mg/kg and MHNA80mg/kg significantly inhibited IL-6production in the abdominal cavity (inhibition rate were48.7%,19.5%and41.2%, respectively).
Part III anti-inflammatory mechanism of MHNA
1, MHNA inhibited NF-κB signaling pathway activation in macrophages
In order to explore the mechanism of MHNA inhibiting macrophage inflammatory response, NF-κB signaling pathway activation was detected by western blot, reporter gene assay and electrophoretic mobility shift assay. Pretreated RAW264.7cells with2.5,5.0,10.0μg/ml MHNA for lh, then cells were cocultured with0.1μg/ml LPS for30min (detection iκB degradation and MAPKs phosphorylation by Western blot) or1h (extracted cytosolic and nuclear proteins, then detected NF-κB distribution in the cytoplasm and nucleus by Western blot and investigated NF-κB DNA binding ability by electrophoretic mobility shift assay.
Western blot results showed that in LPS-stimulated macrophages, iκB protein degradated significantly, and the cytoplasm of NF-κB obviously reduced while NF-κB within the nucleus increased by compared with unstimulated control cells. This indicated that LPS stimulation induced NF-κB translocated from cytoplasm into the nucleus. Pretreatment with MHNA for1h, significantly attenuated the LPS-induced iκB degradation and NF-κB translocation.
Reporter gene results showed that0.1μg/ml LPS caused transcriptional activity of macrophages increased5.3-fold compared to control cells while MHNA significantly inhibited the increasing transcriptional activity of LPS-stimulated macrophages
The results of electrophoretic mobility shift assay showed that LPS increase NF-κB binding activity to its specific DNA sequences in macrophages. Pretreatment macrophages with MHNA and NF-κB specific inhibitor BAY11-7082for1h inhibited the increase of LPS-induced NF-κB and DNA binding activity.
2, MHNA inhibited MAPKs signaling pathway activation in macrophages
RAW264.7cells were pretreated with2.5,5.0,10.0μg/ml MHNA for1h, then cells were cocultured with0.1μg/ml LPS for30min. Total protein was extracted, and MAPKs (p38MAPK, ERK1/2and JNK) phosphorylation were detected by Western blot. The results showed that LPS increased p38MAPK, ERK1/2and JNK phosphorylation in macrophages.2.5-10.Oμg/ml MHNA dose-dependently inhibited p38MAPK and JNK phosphorylation, but had no significant effects on ERK1/2phosphorylation.
Conclusion:
1, MHNA inhibited LPS-induced macrophage inflammatory response.
A, MHNA inhibited inflammatory mediators NO, IL-1β and IL-6production in LPS activated macrophages;
B, MHNA inhibited iNOS and COX-2protein expression in LPS activated macrophages;
C, MHNA inhibited inflammatory genes (iNOS, COX-2, IL-1β and IL-6) expression in LPS activated macrophages.
2, MHNA showed a good anti-inflammatory activity in vivo.
A, MHNA inhibited mouse ear swelling caused by xylene
B, MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.
C, MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis.
3, MHNA inhibited macrophage activation by inhibiting NF-kappa B, p38MAPK and JNK signaling pathway activation.
In summary, MHNA inhibited inflammatory mediators'production in LPS-activated macrophages by blocking NF-κB, p38MAPK and JNK signaling pathway activation. Further studies found that MHNA also showed good anti-inflammtroy activity in xylene-induced mouse ear swelling test and zymosan-induced mouse peritonitis. These studies provide a new experimental basis to investigate the anti-inflammatory activity and mechanism of naphthol derivatives in vitro and in vivo.
引文
[1]Ferrero-Miliani L, Nielsen OH, Andersen PS, Girardin SE. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol.2007.147(2):227-35.
[2]Tonnel AB, Gosset P, Molet S, Tillie-Leblond I, Jeannin P, Joseph M. Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci.1996.796:9-20.
[3]Pasqui AL, Bova G, Maffei S, Auteri A. [Immune factors in atherosclerosis]. Ann Ital Med Int.2005.20(2):81-9.
[4]Nathan C. Points of control in inflammation. Nature.2002.420(6917): 846-52.
[5]李玉林主编.《病理学》第七版.人民卫生出版社.
[6]Ridker PM. C-reactive protein:eighty years from discovery to emergence as a major risk marker for cardiovascular disease. Clin Chem.2009.55(2):209-15.
[7]Kuller LHT, R. P. The role of inflammation in cardiovascular disease.2000. 20(4):901.
[8]Dandona P, Aljada A, Chaudhuri A, Mohanty P. Endothelial dysfunction, inflammation and diabetes. Rev Endocr Metab Disord.2004.5(3):189-97.
[9]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002.420(6917): 860-7.
[10]Akiyama H, Barger S, Barnum S, et al. Inflammation and Alzheimer's disease. Neurobiol Aging.2000.21(3):383-421.
[11]Barbara G, De Giorgio R, Stanghellini V, Cremon C, Corinaldesi R. A role for inflammation in irritable bowel syndrome. Gut.2002.51 Suppl 1:i41-4.
[12]Buchwald D, Wener MH, Pearlman T, Kith P. Markers of inflammation and immune activation in chronic fatigue and chronic fatigue syndrome. J Rheumatol.1997.24(2):372-6.
[13]Sintiprungrat K, Singhto N, Sinchaikul S, Chen ST, Thongboonkerd V. Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate:insights into biological processes. J Proteomics.2010.73(3):602-18.
[14]Douglas SD, Musson RA. Phagocytic defects--monocytes/macrophages. Clin Immunol Immunopathol.1986.40(1):62-8.
[15]Duffield JS. The inflammatory macrophage:a story of Jekyll and Hyde. Clin Sci (Lond).2003.104(1):27-38.
[16]Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011.145(3):341-55.
[17]Wilson HMB, R. N. Erwig LP. Macrophages:promising targets for the treatment of atherosclerosis.2009.7(2):234-43.
[18]Szekanecz Z, Koch AE. Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol.2007.19(3):289-95.
[19]Cutolo MS, A. Barone AS, B. Accardo S. Macrophages, synovial tissue and rheumatoid arthritis.1993.11(3):331-9.
[20]Mahida YR. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease. Inflamm Bowel Dis.2000.6(1):21-33.
[21]West MA, Manthei R, Bubrick MP. Autoregulation of hepatic macrophage activation in sepsis. J Trauma.1993.34(4):473-9; discussion 479-80.
[22]Bogdan C. Nitric oxide and the immune response. Nat Immunol.2001.2(10): 907-16.
[23]Moncada SP, R. M. Higgs EA. Nitric oxide:physiology, pathophysiology, and pharmacology.1991.43(2):109-42.
[24]Tripathi P, Tripathi P, Kashyap L, Singh V. The role of nitric oxide in inflammatory reactions. FEMS Immunol Med Microbiol.2007.51(3):443-52.
[25]Moncada SH, E. A. Endogenous nitric oxide:physiology, pathology and clinical relevance.1991.21(4):361-74.
[26]Moncada SP, R. M. Biosynthesis and actions of nitric oxide.1991.15(1): 16-9.
[27]Kubes P, McCafferty DM. Nitric oxide and intestinal inflammation. Am J Med. 2000.109(2):150-8.
[28]Aktan F. iNOS-mediated nitric oxide production and its regulation.2004. 75(6):639-53.
[29]MacMicking JX, Q. W. Nathan C. Nitric oxide and macrophage function. 1997.15:323-50.
[30]Allen JB, Keng T, Privalle C. Nitric oxide and peroxynitrite production in ocular inflammation. Environ Health Perspect.1998.106 Suppl 5:1145-9.
[31]Nagy G, Clark JM, Buzas El, Gorman CL, Cope AP. Nitric oxide, chronic inflammation and autoimmunity. Immunol Lett.2007.111(1):1-5.
[32]Williams TJ. The role of prostaglandins in inflammation. Ann R Coll Surg Engl.1978.60(3):198-201.
[33]Racis SP, Krysicka-Janniger C. A review of the immunological activities of prostaglandins. Acta Med Pol.1986.27(3-4):177-86.
[34]Botting R. COX-1 and COX-3 inhibitors. Thromb Res.2003.110(5-6): 269-72.
[35]Turini ME, DuBois RN. Cyclooxygenase-2:a therapeutic target. Annu Rev Med.2002.53:35-57.
[36]Seibert K, Zhang Y, Leahy K, et al. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A.1994.91(25):12013-7.
[37]Van Snick J. Interleukin-6:an overview. Annu Rev Immunol.1990.8:253-78.
[38]Braddock M, Quinn A. Targeting IL-1 in inflammatory disease:new opportunities for therapeutic intervention. Nat Rev Drug Discov.2004.3(4): 330-9.
[39]Boraschi DB, P. Macchia GR, P. Tagliabue A. Structure-function relationship in the IL-1 family.1996.1:d270-308.
[40]Sims JES, D. E. The IL-1 family:regulators of immunity.2010.10(2): 89-102.
[41]Nakajima KH, T. [Basic and clinical aspects of IL-6].1991.18(4):505-14.
[42]Ding CC, F. Li JJ, G. Targeting IL-6 in the treatment of inflammatory and autoimmune diseases.2009.18(10):1457-66.
[43]Dendorfer U. Molecular biology of cytokines.1996.20(5):437-44.
[44]Dinarello CA. Proinflammatory cytokines.2000.118(2):503-8.
[45]Feldmann MB, F. M. Maini R. Cytokines in autoimmune disorders.1998. 17(1-4):217-28.
[46]De Benedetti F, Robbioni P, Massa M, Viola S, Albani S, Martini A. Serum interleukin-6 levels and joint involvement in polyarticular and pauciarticular juvenile chronic arthritis. Clin Exp Rheumatol.1992.10(5):493-8.
[47]Hallegua DS, Weisman MH. Potential therapeutic uses of interleukin 1 receptor antagonists in human diseases. Ann Rheum Dis.2002.61(11):960-7.
[48]Molto A, Olive A. Anti-IL-1 molecules:new comers and new indications. Joint Bone Spine.2010.77(2):102-7.
[49]Marsh CB, Wewers MD. The pathogenesis of sepsis. Factors that modulate the response to gram-negative bacterial infection. Clin Chest Med.1996.17(2): 183-97.
[50]Baue AE, Durham R, Faist E. Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF):are we winning the battle. Shock.1998.10(2):79-89.
[51]Martins JO, Ferracini M, Ravanelli N, Landgraf RG, Jancar S. Insulin inhibits LPS-induced signaling pathways in alveolar macrophages. Cell Physiol Biochem.2008.21(4):297-304.
[52]Baldwin AS Jr. Series introduction:the transcription factor NF-kappaB and human disease. J Clin Invest.2001.107(1):3-6.
[53]Pereira SG, Oakley F. Nuclear factor-kappaBl:regulation and function. Int J Biochem Cell Biol.2008.40(8):1425-30.
[54]Beg AA, Baldwin AS Jr. Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor. Oncogene.1994.9(5): 1487-92.
[55]Yamamoto Y, Gaynor RB. IkappaB kinases:key regulators of the NF-kappaB pathway. Trends Biochem Sci.2004.29(2):72-9.
[56]Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol.2009.1(4):a000034.
[57]Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene.1999.18(49):6853-66.
[58]Higuchi T, Watanabe Y, Waga I. Protein disulfide isomerase suppresses the transcriptional activity of NF-kappaB. Biochem Biophys Res Commun.2004. 318(1):46-52.
[59]Takada Y, Singh S, Aggarwal BB. Identification of a p65 peptide that selectively inhibits NF-kappa B activation induced by various inflammatory stimuli and its role in down-regulation of NF-kappaB-mediated gene expression and up-regulation of apoptosis. J Biol Chem.2004.279(15): 15096-104.
[60]Muller JM, Ziegler-Heitbrock HW, Baeuerle PA. Nuclear factor kappa B, a mediator of lipopolysaccharide effects. Immunobiology.1993.187(3-5): 233-56.
[61]Sharif O, Bolshakov VN, Raines S, Newham P, Perkins ND. Transcriptional profiling of the LPS induced NF-kappaB response in macrophages. BMC Immunol.2007.8:1.
[62]Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol.2002.2(10):725-34.
[63]Su B, Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol.1996.8(3):402-11.
[64]Zhang YL, Dong C. MAP kinases in immune responses. Cell Mol Immunol. 2005.2(1):20-7.
[65]Rao KM. MAP kinase activation in macrophages. J Leukoc Biol.2001.69(1): 3-10.
[66]Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol.1996.8(2):205-15.
[67]杨宗炎.非甾体抗炎药市场分析.中国制药信息.2000.16(3)：33-39.
[68]徐叔云,魏伟.我国抗炎免疫药理研究现状.中国药理学通报.1992.8(3)：161-165.
[69]Frey FJ. [Practical aspects of glucocorticosteroid therapy]. Schweiz Med Wochenschr.1984.114(11):354-60.
[70]谢宝官.非甾类抗炎药物的合理使用.解放军医学情报.1995.9(6)：288-291.
[71]McKanna JA, Zhang MZ, Wang JL, Cheng H, Harris RC. Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am J Physiol.1998. 275(1 Pt 2):R227-33.
[72]Khorana MLP, S. Y. Pishawikar AD. Antibacterial and antifungal properties of beta-naphthol derivatives. VI.1967.56(8):993-7.
[73]Pandolfo LP, F. [Action of naphthalene and some of its derivatives on anaerobic glycolysis. II. Action of naphthalene, beta-naphthol and beta-naphthylamine on glycolysis of some animal tissues].1957.33(10-11): 1794-5.
[74]Vanlerenberghe JG, F. Milbled GR, A. [Choleretic activity of some derivatives of the 1-naphthol series].1960.154:728-32.
[75]Cohen GMW, G. D. Gibby EMS, M. T. Doherty MDC, T. A.1-Naphthol:a potential selective antitumour agent.1983.32(15):2363-5.
[76]Jones MdD, M. Cohen GM. Antitumour activity of 1-naphthol against L1210 leukaemia in vivo and Ehrlich ascites tumour cells in vivo and in vitro.1986. 33(3):347-54.
[77]Wilson GDdD, M. Cohen GM. Selective toxicity of 1-naphthol to human colorectal tumour tissue.1985.51(6):853-63.
[78]Urbahns KY, T. Mogi MT, M. Fujishima HM, et al. Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence.2011.21(11): 3354-7.
[79]Kongkathip N, Hasitapan K, Pradidphol N, Kirtikara K, Jongkona N, Kongkathip B. Synthesis of novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives with anticyclooxygenase activity. Curr Med Chem.2006.13(30):3663-74.
[80]Kongkathip B, Sangma C, Kirtikara K, et al. Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II. Bioorg Med Chem.2005.13(6):2167-75.
[81]Arenberger PR, A. Armah BK, L. Ruzicka T. The lipoxygenase inhibitor 2-phenylmethyl-1-naphthol (DuP 654) is a 12(S)-hydroxyeicosatetraenoic acid receptor antagonist in the human epidermal cell line SCL-II.1993.6(2): 148-51.
[82]Matsubara M, Yamachika E, Tsujigiwa H, et al. Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption. Osteoporos Int.2010.21(8):1437-47.
[83]Okada Y, Tsuzuki Y, Miyazaki J, et al. Propionibacterium freudenreichii component 1.4-dihydroxy-2-naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing. Gut.2006.55(5):681-8.
[84]Cross RK, Wilson KT. Nitric oxide in inflammatory bowel disease. Inflamm Bowel Dis.2003.9(3):179-89.
[85]Nagy G, Koncz A, Telarico T, et al. Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther.2010.12(3):210.
[86]Di RM, Radomski M, Carnuccio R, Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochem Biophys Res Commun.1990.172(3):1246-52.
[87]Tang X, Marciano DL, Leeman SE, Amar S. LPS induces the interaction of a transcription factor, LPS-induced TNF-alpha factor, and STAT6(B) with effects on multiple cytokines. Proc Natl Acad Sci U S A.2005.102(14): 5132-7.
[88]Ryan GB, Majno G. Acute inflammation. A review. Am J Pathol.1977.86(1): 183-276.
[89]Liu J, Yang F, Zhang Y, Li J. Studies on the cell-immunosuppressive mechanism of Oridonin from Isodon serra. Int Immunopharmacol.2007.7(7): 945-54.
[90]Feng X, Cheng G, Chen S Y, Yang H, Huang W. Evaluation of the burn healing properties of oil extraction from housefly larva in mice. J Ethnopharmacol. 2010.130(3):586-92.
[91]Leite DF, Echevarria-Lima J, Ferreira SC, Calixto JB, Rumjanek VM. ABCC transporter inhibition reduces zymosan-induced peritonitis. J Leukoc Biol. 2007.82(3):630-7.
[92]Mikasa K, Kita E, Sawaki M, et al. The anti-inflammatory effect of erythromycin in zymosan-induced peritonitis of mice. J Antimicrob Chemother.1992.30(3):339-48.
[93]Gado KG, G. Zymosan inflammation:a new method suitable for evaluating new antiinflammatory drugs.1991.32(1-2):119-21.
[94]Kolaczkowska E, Arnold B, Opdenakker G. Gelatinase B/MMP-9 as an inflammatory marker enzyme in mouse zymosan peritonitis:comparison of phase-specific and cell-specific production by mast cells, macrophages and neutrophils. Immunobiology.2008.213(2):109-24.
[95]Pan Q, Liu Y, Zheng J, et al. Protective effect of chloral hydrate against lipopolysaccharide/D-galactosamine-induced acute lethal liver injury and zymosan-induced peritonitis in mice. Int Immunopharmacol.2010.
[96]Rao TS, Currie JL, Shaffer AF, Isakson PC. In vivo characterization of zymosan-induced mouse peritoneal inflammation. J Pharmacol Exp Ther. 1994.269(3):917-25.
[97]Underhill DM. Macrophage recognition of zymosan particles.2003.9(3): 176-80.
[98]Caamano J, Hunter CA. NF-kappaB family of transcription factors:central regulators of innate and adaptive immune functions. Clin Microbiol Rev.2002. 15(3):414-29.
[99]Kiemer AK, Hartung T, Huber C, Vollmar AM. Phyllanthus amarus has anti-inflammatory potential by inhibition of iNOS, COX-2, and cytokines via the NF-kappaB pathway. J Hepatol.2003.38(3):289-97.
[100]Makarov SS. NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today.2000.6(11):441-8.
[101]Hiransai P, Ratanachaiyavong S, Itharat A, Graidist P, Ruengrairatanaroj P, Purintrapiban J. Dioscorealide B suppresses LPS-induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF-kappaB and ERK1/2 activation. J Cell Biochem.2010. 109(5):1057-63.
[102]Jung HJ, Kim SJ, Jeon WK, et al. Anti-inflammatory activity of n-propyl gallate through down-regulation of NF-kappaB and JNK pathways. Inflammation.2011.34(5):352-61.
[103]Mendis E, Kim MM, Rajapakse N, Kim SK. Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions. Bioorg Med Chem.2008.16(18):8390-6.
[104]Emery JGO, E. H. Jaye M. Therapeutic modulation of transcription factor activity.2001.22(5):233-40.
[105]Barnes PJ. Anti-inflammatory actions of glucocorticoids:molecular mechanisms.1998.94(6):557-72.
[106]Yamamoto YY, M. J. Lin KMG, R. B. Sulindac inhibits activation of the NF-kappaB pathway.1999.274(38):27307-14.
[107]Yin MJY, Y. Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta.1998.396(6706):77-80.
[108]Chaturvedi MMK, A. Darnay BGC, G. B. Agarwal SA, B. B. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kappaB activation, IkappaBalpha phosphorylation, and degradation.1997.272(48):30129-34.
[109]Huang GS, L. Z. Chi H. Regulation of JNK and p38 MAPK in the immune system:signal integration, propagation and termination.2009.48(3):161-9.
[110]Roy PKR, F. Bragg JI, J. A. Role of the JNK signal transduction pathway in inflammatory bowel disease.2008.14(2):200-2.
[111]Herlaar EB, Z. p38 MAPK signalling cascades in inflammatory disease.1999. 5(10):439-47.
[112]Caelles CG, J. M. Munoz A. Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway.1997.11(24):3351-64.
[113]Gupta SB, T. Whitmarsh AJC, J. Sluss HKD, B. Davis RJ. Selective interaction of JNK protein kinase isoforms with transcription factors.1996. 15(11):2760-70.
[114]Fukuda KK, A. Watabe K. The role of tumor-associated macrophage in tumor progression.2012.4:787-98.
[115]Hagemann T, Biswas SK, Lawrence T, Sica A, Lewis CE. Regulation of macrophage function in tumors:the multifaceted role of NF-kappaB. Blood. 2009.113(14):3139-46.
[116]Nardin A, Abastado JP. Macrophages and cancer. Front Biosci.2008.13: 3494-505.
[117]Allavena P, Garlanda C, Borrello MG, Sica A, Mantovani A. Pathways connecting inflammation and cancer. Curr Opin Genet Dev.2008.18(1):3-10.
[118]Zhang Z, Rigas B. NF-kappaB, inflammation and pancreatic carcinogenesis: NF-kappaB as a chemoprevention target (review). Int J Oncol.2006.29(1): 185-92.
[119]Biswas SK, Lewis CE. NF-kappaB as a central regulator of macrophage function in tumors. J Leukoc Biol.2010.88(5):877-84.
[2]Tonnel AB, Gosset P, Molet S, Tillie-Leblond I, Jeannin P, Joseph M. Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci.1996.796:9-20.
[3]Pasqui AL, Bova G, Maffei S, Auteri A. [Immune factors in atherosclerosis]. Ann Ital Med Int.2005.20(2):81-9.
[4]Nathan C. Points of control in inflammation. Nature.2002.420(6917): 846-52.
[5]李玉林主编.《病理学》第七版.人民卫生出版社.
[6]Ridker PM. C-reactive protein:eighty years from discovery to emergence as a major risk marker for cardiovascular disease. Clin Chem.2009.55(2):209-15.
[7]Kuller LHT, R. P. The role of inflammation in cardiovascular disease.2000. 20(4):901.
[8]Dandona P, Aljada A, Chaudhuri A, Mohanty P. Endothelial dysfunction, inflammation and diabetes. Rev Endocr Metab Disord.2004.5(3):189-97.
[9]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002.420(6917): 860-7.
[10]Akiyama H, Barger S, Barnum S, et al. Inflammation and Alzheimer's disease. Neurobiol Aging.2000.21(3):383-421.
[11]Barbara G, De Giorgio R, Stanghellini V, Cremon C, Corinaldesi R. A role for inflammation in irritable bowel syndrome. Gut.2002.51 Suppl 1:i41-4.
[12]Buchwald D, Wener MH, Pearlman T, Kith P. Markers of inflammation and immune activation in chronic fatigue and chronic fatigue syndrome. J Rheumatol.1997.24(2):372-6.
[13]Sintiprungrat K, Singhto N, Sinchaikul S, Chen ST, Thongboonkerd V. Alterations in cellular proteome and secretome upon differentiation from monocyte to macrophage by treatment with phorbol myristate acetate:insights into biological processes. J Proteomics.2010.73(3):602-18.
[14]Douglas SD, Musson RA. Phagocytic defects--monocytes/macrophages. Clin Immunol Immunopathol.1986.40(1):62-8.
[15]Duffield JS. The inflammatory macrophage:a story of Jekyll and Hyde. Clin Sci (Lond).2003.104(1):27-38.
[16]Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011.145(3):341-55.
[17]Wilson HMB, R. N. Erwig LP. Macrophages:promising targets for the treatment of atherosclerosis.2009.7(2):234-43.
[18]Szekanecz Z, Koch AE. Macrophages and their products in rheumatoid arthritis. Curr Opin Rheumatol.2007.19(3):289-95.
[19]Cutolo MS, A. Barone AS, B. Accardo S. Macrophages, synovial tissue and rheumatoid arthritis.1993.11(3):331-9.
[20]Mahida YR. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease. Inflamm Bowel Dis.2000.6(1):21-33.
[21]West MA, Manthei R, Bubrick MP. Autoregulation of hepatic macrophage activation in sepsis. J Trauma.1993.34(4):473-9; discussion 479-80.
[22]Bogdan C. Nitric oxide and the immune response. Nat Immunol.2001.2(10): 907-16.
[23]Moncada SP, R. M. Higgs EA. Nitric oxide:physiology, pathophysiology, and pharmacology.1991.43(2):109-42.
[24]Tripathi P, Tripathi P, Kashyap L, Singh V. The role of nitric oxide in inflammatory reactions. FEMS Immunol Med Microbiol.2007.51(3):443-52.
[25]Moncada SH, E. A. Endogenous nitric oxide:physiology, pathology and clinical relevance.1991.21(4):361-74.
[26]Moncada SP, R. M. Biosynthesis and actions of nitric oxide.1991.15(1): 16-9.
[27]Kubes P, McCafferty DM. Nitric oxide and intestinal inflammation. Am J Med. 2000.109(2):150-8.
[28]Aktan F. iNOS-mediated nitric oxide production and its regulation.2004. 75(6):639-53.
[29]MacMicking JX, Q. W. Nathan C. Nitric oxide and macrophage function. 1997.15:323-50.
[30]Allen JB, Keng T, Privalle C. Nitric oxide and peroxynitrite production in ocular inflammation. Environ Health Perspect.1998.106 Suppl 5:1145-9.
[31]Nagy G, Clark JM, Buzas El, Gorman CL, Cope AP. Nitric oxide, chronic inflammation and autoimmunity. Immunol Lett.2007.111(1):1-5.
[32]Williams TJ. The role of prostaglandins in inflammation. Ann R Coll Surg Engl.1978.60(3):198-201.
[33]Racis SP, Krysicka-Janniger C. A review of the immunological activities of prostaglandins. Acta Med Pol.1986.27(3-4):177-86.
[34]Botting R. COX-1 and COX-3 inhibitors. Thromb Res.2003.110(5-6): 269-72.
[35]Turini ME, DuBois RN. Cyclooxygenase-2:a therapeutic target. Annu Rev Med.2002.53:35-57.
[36]Seibert K, Zhang Y, Leahy K, et al. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A.1994.91(25):12013-7.
[37]Van Snick J. Interleukin-6:an overview. Annu Rev Immunol.1990.8:253-78.
[38]Braddock M, Quinn A. Targeting IL-1 in inflammatory disease:new opportunities for therapeutic intervention. Nat Rev Drug Discov.2004.3(4): 330-9.
[39]Boraschi DB, P. Macchia GR, P. Tagliabue A. Structure-function relationship in the IL-1 family.1996.1:d270-308.
[40]Sims JES, D. E. The IL-1 family:regulators of immunity.2010.10(2): 89-102.
[41]Nakajima KH, T. [Basic and clinical aspects of IL-6].1991.18(4):505-14.
[42]Ding CC, F. Li JJ, G. Targeting IL-6 in the treatment of inflammatory and autoimmune diseases.2009.18(10):1457-66.
[43]Dendorfer U. Molecular biology of cytokines.1996.20(5):437-44.
[44]Dinarello CA. Proinflammatory cytokines.2000.118(2):503-8.
[45]Feldmann MB, F. M. Maini R. Cytokines in autoimmune disorders.1998. 17(1-4):217-28.
[46]De Benedetti F, Robbioni P, Massa M, Viola S, Albani S, Martini A. Serum interleukin-6 levels and joint involvement in polyarticular and pauciarticular juvenile chronic arthritis. Clin Exp Rheumatol.1992.10(5):493-8.
[47]Hallegua DS, Weisman MH. Potential therapeutic uses of interleukin 1 receptor antagonists in human diseases. Ann Rheum Dis.2002.61(11):960-7.
[48]Molto A, Olive A. Anti-IL-1 molecules:new comers and new indications. Joint Bone Spine.2010.77(2):102-7.
[49]Marsh CB, Wewers MD. The pathogenesis of sepsis. Factors that modulate the response to gram-negative bacterial infection. Clin Chest Med.1996.17(2): 183-97.
[50]Baue AE, Durham R, Faist E. Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF):are we winning the battle. Shock.1998.10(2):79-89.
[51]Martins JO, Ferracini M, Ravanelli N, Landgraf RG, Jancar S. Insulin inhibits LPS-induced signaling pathways in alveolar macrophages. Cell Physiol Biochem.2008.21(4):297-304.
[52]Baldwin AS Jr. Series introduction:the transcription factor NF-kappaB and human disease. J Clin Invest.2001.107(1):3-6.
[53]Pereira SG, Oakley F. Nuclear factor-kappaBl:regulation and function. Int J Biochem Cell Biol.2008.40(8):1425-30.
[54]Beg AA, Baldwin AS Jr. Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor. Oncogene.1994.9(5): 1487-92.
[55]Yamamoto Y, Gaynor RB. IkappaB kinases:key regulators of the NF-kappaB pathway. Trends Biochem Sci.2004.29(2):72-9.
[56]Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol.2009.1(4):a000034.
[57]Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene.1999.18(49):6853-66.
[58]Higuchi T, Watanabe Y, Waga I. Protein disulfide isomerase suppresses the transcriptional activity of NF-kappaB. Biochem Biophys Res Commun.2004. 318(1):46-52.
[59]Takada Y, Singh S, Aggarwal BB. Identification of a p65 peptide that selectively inhibits NF-kappa B activation induced by various inflammatory stimuli and its role in down-regulation of NF-kappaB-mediated gene expression and up-regulation of apoptosis. J Biol Chem.2004.279(15): 15096-104.
[60]Muller JM, Ziegler-Heitbrock HW, Baeuerle PA. Nuclear factor kappa B, a mediator of lipopolysaccharide effects. Immunobiology.1993.187(3-5): 233-56.
[61]Sharif O, Bolshakov VN, Raines S, Newham P, Perkins ND. Transcriptional profiling of the LPS induced NF-kappaB response in macrophages. BMC Immunol.2007.8:1.
[62]Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol.2002.2(10):725-34.
[63]Su B, Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol.1996.8(3):402-11.
[64]Zhang YL, Dong C. MAP kinases in immune responses. Cell Mol Immunol. 2005.2(1):20-7.
[65]Rao KM. MAP kinase activation in macrophages. J Leukoc Biol.2001.69(1): 3-10.
[66]Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol.1996.8(2):205-15.
[67]杨宗炎.非甾体抗炎药市场分析.中国制药信息.2000.16(3)：33-39.
[68]徐叔云,魏伟.我国抗炎免疫药理研究现状.中国药理学通报.1992.8(3)：161-165.
[69]Frey FJ. [Practical aspects of glucocorticosteroid therapy]. Schweiz Med Wochenschr.1984.114(11):354-60.
[70]谢宝官.非甾类抗炎药物的合理使用.解放军医学情报.1995.9(6)：288-291.
[71]McKanna JA, Zhang MZ, Wang JL, Cheng H, Harris RC. Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am J Physiol.1998. 275(1 Pt 2):R227-33.
[72]Khorana MLP, S. Y. Pishawikar AD. Antibacterial and antifungal properties of beta-naphthol derivatives. VI.1967.56(8):993-7.
[73]Pandolfo LP, F. [Action of naphthalene and some of its derivatives on anaerobic glycolysis. II. Action of naphthalene, beta-naphthol and beta-naphthylamine on glycolysis of some animal tissues].1957.33(10-11): 1794-5.
[74]Vanlerenberghe JG, F. Milbled GR, A. [Choleretic activity of some derivatives of the 1-naphthol series].1960.154:728-32.
[75]Cohen GMW, G. D. Gibby EMS, M. T. Doherty MDC, T. A.1-Naphthol:a potential selective antitumour agent.1983.32(15):2363-5.
[76]Jones MdD, M. Cohen GM. Antitumour activity of 1-naphthol against L1210 leukaemia in vivo and Ehrlich ascites tumour cells in vivo and in vitro.1986. 33(3):347-54.
[77]Wilson GDdD, M. Cohen GM. Selective toxicity of 1-naphthol to human colorectal tumour tissue.1985.51(6):853-63.
[78]Urbahns KY, T. Mogi MT, M. Fujishima HM, et al. Naphthol derivatives as TRPV1 inhibitors for the treatment of urinary incontinence.2011.21(11): 3354-7.
[79]Kongkathip N, Hasitapan K, Pradidphol N, Kirtikara K, Jongkona N, Kongkathip B. Synthesis of novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives with anticyclooxygenase activity. Curr Med Chem.2006.13(30):3663-74.
[80]Kongkathip B, Sangma C, Kirtikara K, et al. Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II. Bioorg Med Chem.2005.13(6):2167-75.
[81]Arenberger PR, A. Armah BK, L. Ruzicka T. The lipoxygenase inhibitor 2-phenylmethyl-1-naphthol (DuP 654) is a 12(S)-hydroxyeicosatetraenoic acid receptor antagonist in the human epidermal cell line SCL-II.1993.6(2): 148-51.
[82]Matsubara M, Yamachika E, Tsujigiwa H, et al. Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption. Osteoporos Int.2010.21(8):1437-47.
[83]Okada Y, Tsuzuki Y, Miyazaki J, et al. Propionibacterium freudenreichii component 1.4-dihydroxy-2-naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing. Gut.2006.55(5):681-8.
[84]Cross RK, Wilson KT. Nitric oxide in inflammatory bowel disease. Inflamm Bowel Dis.2003.9(3):179-89.
[85]Nagy G, Koncz A, Telarico T, et al. Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther.2010.12(3):210.
[86]Di RM, Radomski M, Carnuccio R, Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochem Biophys Res Commun.1990.172(3):1246-52.
[87]Tang X, Marciano DL, Leeman SE, Amar S. LPS induces the interaction of a transcription factor, LPS-induced TNF-alpha factor, and STAT6(B) with effects on multiple cytokines. Proc Natl Acad Sci U S A.2005.102(14): 5132-7.
[88]Ryan GB, Majno G. Acute inflammation. A review. Am J Pathol.1977.86(1): 183-276.
[89]Liu J, Yang F, Zhang Y, Li J. Studies on the cell-immunosuppressive mechanism of Oridonin from Isodon serra. Int Immunopharmacol.2007.7(7): 945-54.
[90]Feng X, Cheng G, Chen S Y, Yang H, Huang W. Evaluation of the burn healing properties of oil extraction from housefly larva in mice. J Ethnopharmacol. 2010.130(3):586-92.
[91]Leite DF, Echevarria-Lima J, Ferreira SC, Calixto JB, Rumjanek VM. ABCC transporter inhibition reduces zymosan-induced peritonitis. J Leukoc Biol. 2007.82(3):630-7.
[92]Mikasa K, Kita E, Sawaki M, et al. The anti-inflammatory effect of erythromycin in zymosan-induced peritonitis of mice. J Antimicrob Chemother.1992.30(3):339-48.
[93]Gado KG, G. Zymosan inflammation:a new method suitable for evaluating new antiinflammatory drugs.1991.32(1-2):119-21.
[94]Kolaczkowska E, Arnold B, Opdenakker G. Gelatinase B/MMP-9 as an inflammatory marker enzyme in mouse zymosan peritonitis:comparison of phase-specific and cell-specific production by mast cells, macrophages and neutrophils. Immunobiology.2008.213(2):109-24.
[95]Pan Q, Liu Y, Zheng J, et al. Protective effect of chloral hydrate against lipopolysaccharide/D-galactosamine-induced acute lethal liver injury and zymosan-induced peritonitis in mice. Int Immunopharmacol.2010.
[96]Rao TS, Currie JL, Shaffer AF, Isakson PC. In vivo characterization of zymosan-induced mouse peritoneal inflammation. J Pharmacol Exp Ther. 1994.269(3):917-25.
[97]Underhill DM. Macrophage recognition of zymosan particles.2003.9(3): 176-80.
[98]Caamano J, Hunter CA. NF-kappaB family of transcription factors:central regulators of innate and adaptive immune functions. Clin Microbiol Rev.2002. 15(3):414-29.
[99]Kiemer AK, Hartung T, Huber C, Vollmar AM. Phyllanthus amarus has anti-inflammatory potential by inhibition of iNOS, COX-2, and cytokines via the NF-kappaB pathway. J Hepatol.2003.38(3):289-97.
[100]Makarov SS. NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today.2000.6(11):441-8.
[101]Hiransai P, Ratanachaiyavong S, Itharat A, Graidist P, Ruengrairatanaroj P, Purintrapiban J. Dioscorealide B suppresses LPS-induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF-kappaB and ERK1/2 activation. J Cell Biochem.2010. 109(5):1057-63.
[102]Jung HJ, Kim SJ, Jeon WK, et al. Anti-inflammatory activity of n-propyl gallate through down-regulation of NF-kappaB and JNK pathways. Inflammation.2011.34(5):352-61.
[103]Mendis E, Kim MM, Rajapakse N, Kim SK. Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions. Bioorg Med Chem.2008.16(18):8390-6.
[104]Emery JGO, E. H. Jaye M. Therapeutic modulation of transcription factor activity.2001.22(5):233-40.
[105]Barnes PJ. Anti-inflammatory actions of glucocorticoids:molecular mechanisms.1998.94(6):557-72.
[106]Yamamoto YY, M. J. Lin KMG, R. B. Sulindac inhibits activation of the NF-kappaB pathway.1999.274(38):27307-14.
[107]Yin MJY, Y. Gaynor RB. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta.1998.396(6706):77-80.
[108]Chaturvedi MMK, A. Darnay BGC, G. B. Agarwal SA, B. B. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kappaB activation, IkappaBalpha phosphorylation, and degradation.1997.272(48):30129-34.
[109]Huang GS, L. Z. Chi H. Regulation of JNK and p38 MAPK in the immune system:signal integration, propagation and termination.2009.48(3):161-9.
[110]Roy PKR, F. Bragg JI, J. A. Role of the JNK signal transduction pathway in inflammatory bowel disease.2008.14(2):200-2.
[111]Herlaar EB, Z. p38 MAPK signalling cascades in inflammatory disease.1999. 5(10):439-47.
[112]Caelles CG, J. M. Munoz A. Nuclear hormone receptor antagonism with AP-1 by inhibition of the JNK pathway.1997.11(24):3351-64.
[113]Gupta SB, T. Whitmarsh AJC, J. Sluss HKD, B. Davis RJ. Selective interaction of JNK protein kinase isoforms with transcription factors.1996. 15(11):2760-70.
[114]Fukuda KK, A. Watabe K. The role of tumor-associated macrophage in tumor progression.2012.4:787-98.
[115]Hagemann T, Biswas SK, Lawrence T, Sica A, Lewis CE. Regulation of macrophage function in tumors:the multifaceted role of NF-kappaB. Blood. 2009.113(14):3139-46.
[116]Nardin A, Abastado JP. Macrophages and cancer. Front Biosci.2008.13: 3494-505.
[117]Allavena P, Garlanda C, Borrello MG, Sica A, Mantovani A. Pathways connecting inflammation and cancer. Curr Opin Genet Dev.2008.18(1):3-10.
[118]Zhang Z, Rigas B. NF-kappaB, inflammation and pancreatic carcinogenesis: NF-kappaB as a chemoprevention target (review). Int J Oncol.2006.29(1): 185-92.
[119]Biswas SK, Lewis CE. NF-kappaB as a central regulator of macrophage function in tumors. J Leukoc Biol.2010.88(5):877-84.