胡小鹏，金 哲，贺震旦

深圳大学医学部医学院药学系 深圳大学生物治疗研究所 深圳天然小分子创新药物工程实验室，深圳 518060

中医药具有五千多年的历史，国内外科学家对中药、天然药、民间药和民族药物进行了大量的研究，具有抗病毒药用植物约100 余种［1］，如金银花(Lonicera japonica)、黄芩(Scutellaria baicalensis)、黄芪(Astragalus membranaceus)、五味子(Schisandra chinensis(Turcz.))、夏枯草(Prunella vulgaris)、连翘(Forsythia suspensa)等(见表1)，据迄今为止的研究表明天然小分子(Natural micromolecule)为这些抗病毒药用植物活性的物质基础。

抗病毒细胞因子(Antiviral cytokines)曾在Nasu K［2］和Fakruddin JM［3］等人发表的学术论文中提及过，但未曾对其作出具体的定义。为系统地开展细胞因子抗病毒作用及其作用机制研究的研究，有必要对抗病毒细胞因子进行明确地定义:抗病毒细胞因子是由机体细胞分泌的，具有联动机体固有免疫和特异性免疫应答，进而抑制或捕杀体内病毒的小分子蛋白。抗病毒细胞因子可分两大类:一类是可直接作用体细胞产生抗病毒蛋白，即1)干扰素(Interferon):IFN-α、IFN-β、IFN-γ 和IFN-λ(IL-28、IL-29)［4-7］;2)肿 瘤 坏 死 因 子(Tumor Necrosis Factor)［8］:TNF-α 和TNF-β;干扰素和肿瘤坏死因子可直接激活细胞内JAK(Janus activated kinase)-STAT(signal transducer and activator of transcription)信号通路［9-13］，随后引起下游信号级联反应，包括MTOR信号通路、PI3K 信号通路、MAPK 信号通路均被激活，最终启动细胞核中一些关键的蛋白基因进行转录，翻译出抗病毒蛋白进而对细菌、病毒以及癌细胞等产生防御作用［14］(见图1)。另一类是能激活巨噬细胞、自然杀伤细胞(NK 细胞)和毒性T 淋巴细胞(CTL)等细胞，进而捕杀或吞噬体内病毒的细胞因子(见图1)，即白介素(Interleukin):IL-1β、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-21、IL-27等［15-20］。

基于细胞因子对捕杀或抑制病毒方面的作用，本文综述了国内外迄今为止报道的可诱生抗病毒细胞因子的天然小分子(见图2，图3)，其中以多酚类、苷类居多，还有部分物质基础未明确的提取物，有待进一步研究。

1 多酚类化合物(Polyphenolic compounds)

白藜芦醇(1，Resveratrol，10 mol/L)能上调人外周血单核细胞IL-1β、IL-6、TNF-α，调节免疫细胞功能［21］。从姜黄(Curcuma longa)根茎分离的天然多酚姜黄素(2，Curcumin)，通过激活人细胞内MAPK以及MTOR 等信号通路，调节下游STATs 蛋白磷酸化水平，控制TNF-α、IL-1 和IL-6 的表达分泌［22］，姜黄素还可以显著的调控TRAIL(TNF 相关凋亡诱导配基)，有研究显示姜黄素(10 μmol/L 和25 μmol/L)和TRAIL(20 ng/mL)联合使用能够促进胞内半胱天冬酶的前体Procaspase-3，8，9 的分裂，并使得线粒体释放细胞色素C，导致细胞毒性的增强［23］。茶多酚复方制剂(50～150 mg/kg)可增强小鼠淋巴细胞增殖能力，提高血清溶菌酶活性，上调血清中IFN-γ 分泌［24］。番茄(Lycopersicum esculentum)的多酚提取物能够调节牛中性粒细胞TNF-α 基因的表达，加热处理的提取物可显著上调IFN-γ 的分泌［25］，具有增强动物和人类固有免疫的潜在活性。由咖啡酸(3，Caffeic acid)、阿魏酸(4，Ferulic acid)和对香豆酸(5，p-Coumaric acid)酶促聚合的多酚(20～100 μg/mL)，通过和T 细胞表面CD4+蛋白结合，激活下游信号通路后上调小鼠脾脏细胞中的IFN-γ 和GM-CSF(Granulocyte-macrophage Colonystimulating Factor)分泌量［26］，增强机体固有免疫。

从Cocoa 提取的黄酮类化合物，能够减少小鼠腹膜巨噬细胞活性氧和NO 的生成，上调IFN-γ 和TNF-α 的作用［27］。中药黄芪(Astragalus membranaceus)和黄芩(Scutellaria baicalensis)是传统的抗病毒药材，主含槲皮素(6，Quercetin)、黄芩素(7，Baicalein)等天然小分子。Nair［28］等人报道，槲皮素(5 μmol/L)可调节外周血单核细胞IFN-γ 和IL-4 的分泌，从而促使Th0 细胞向Th1 细胞分化;随后进行了攻毒试验，发现诱生的IFN-γ 具有很强的抗VSV(vesicular stomatitis virus)活性。Johari 等［29］报道槲皮素和黄芩素抑制JEV(Japanese Encephalitis Virus)的IC50分别为212.1、14.28 μg/mL，提示黄芩素比槲皮素具有更好的抗JEV 作用。有研究表明，黄芩素可直接抑制病毒血凝素和神经氨酸酶［30］。

桂皮醛(13，Cinnamaldehyde，1 mg/kg)［31，32］可上调小鼠血清中IL-2、IL-10 的分泌量，其诱生机制可能与Toll 样受体的激活有关。从百屈菜中提取得到 血 根 碱(14，6-acetonyl-5，6-dihydrosanguinarine)［33］，体外实验证实其可诱导巨噬细胞和树突状细胞分泌TFN-α、IL-6、IL-8 等抗病毒细胞因子，对其诱生机制的研究表明，血根碱可激活MAPK 通路中ERK，NF-kB 循环活化机制也参与其中，共同调控了下游抗病毒细胞因子的表达。朱晓新等人［34］从紫萁贯众分离得到四种多酚化合物:4＇-hydroxybenzalacetone(HBAc)，3＇，4＇-dihydroxybenzalacetone(DHBAc)、4-hydroxybenzaldehyde(HBAI)、3，4-dihydroxybenzaldehyde(DHBAI)，其中HBAI(15，10-100 ug/mL)、DHBAI(16，10-100 ug/mL)可激活胞内NF-kB 通路，可显著上调小鼠腹膜细胞分泌TNF、IL-1β、IFN-γ 等，诱生机制可能和iNOS(一氧化氮合成酶)的激活和NO 的生成有关。

2 苷类化合物(Glycosides)

研究表明苷类化合物具有抗肿瘤抗病毒活性［35］;桔梗(Platycodon grandiflorum)中分离得到的单体桔梗苷D(8，Platycodin D，0.0016～0.2 μg/mL)［36］，能够上调IL-2、IFN-γ 的mRNA 表达水平，调控Th1/Th2 细胞的转录因子T-bet 和CATA-3 表达，同时，桔梗苷D 可以提高OVA 致敏小鼠抗体水平，具有免疫调节作用。王先远等［37］研究苦瓜总皂苷对衰老小鼠免疫功能的影响，发现苦瓜总皂苷不仅可上调血清中IL-2 水平，而且促进脾脏分泌IL-2及腹腔巨噬细胞分泌TNF-α，其机制可能与DNA 多聚酶II 活性增强有关;苦瓜总皂苷促进IL-2 分泌，改变T 细胞亚群的组成，使机体免疫状态趋向年轻化。徐先祥等［38］人报道甘草皂苷(9，0.5 mg/mL)可抑制98%以上的HIV 增殖，0.125 mg/mL 时，可抑制98%以上的HIV 的增殖，并可抑制50%斑块的形成，其作用机制是诱生干扰素和增强NK 细胞的活性抵抗HIV。柚皮苷(17，naringin，20，40 and 80 mg/kg)［39］可上调Th2 型细胞分泌IL-4、IL-6、IL-10等。

表1 抗病毒药用植物Table 1 Antivial Medical Plants

注:* 已报道的可诱生抗病毒细胞因子的天然小分子Note:* These natural micromolecules have been reported to induce antiviral cytokines.

图1 天然小分子诱生细胞因子抗病毒作用机制Fig.1 The mechanisms of antiviral activity of antiviral cytokines induced by natrual micromolecule

图2 诱生抗病毒细胞因子的天然小分子1～12 的化学结构式Fig.2 The chemical structure of natural micromolecule 1-12 with the function of inducing antiviral cytokines

图3 诱生抗病毒细胞因子的天然小分子13～19 的化学结构式Fig.3 The chemical structure of natural micromolecule 13-19 with the function of inducing antiviral cytokines

另外，本课题组开展天然小分子诱生机体抗病毒细胞因子的研究中，发现天麻素(10，Gastrodin，0～1000 μmol/L)、迎春花素(11，Jasminin，1-100 μmol/L)能够显著上调RAW246.7 细胞分泌TNFα;从苦丁茶紫茎女贞(Ligustrum purpurascens)叶提取得到苯丙素总苷(crude phenylethanoid glycosides)，属多酚类化合物，活性测试表明，苯丙素总苷(0.22～1.32 g/kg)具有免疫增强作用［49］;进一步研究还发现，从苯丙素总苷分离得到的单体阿克苷(12，acteoside，0.8～200 μg/mL)，能够显著上调Balb/c 小鼠体内IFN-γ 的表达水平，动物攻毒实验证实可显著抑制小鼠体内病毒载量。

3 其他

大蒜辣素(18，Allitridin，25 mg/kg/day)［40］可调节CMV 感染小鼠体内Foxp3 蛋白表达，上调机体IFN-γ 和IL-4 分泌，恢复CMV 攻击造成的免疫系统紊乱。青蒿素(19，Artemisinin)［41］可显著刺激巨噬细胞分泌IL-12，其诱生机制和JNK 磷酸化的抑制有关。

滑菇(Pholiota nameko)提取物(20～60 mg/kg)［42］能够上调白介素、肿瘤坏死因子、干扰素等多种抗病毒细胞因子的分泌。

此外，许多中药提取物具有免疫增强作用［43］，如黄芪提取物(100～200 μg/mL)、板蓝根提取物(100～200 μg/mL)能够刺激鸡外周血淋巴细胞IFN-γ 的mRNA 的表达量，并对NDV(Newcastle disease virus)感染有抑制作用。人参叶提取物［44］可显著上调感染H5N1 小鼠和雪貂肺部IFN-γ 分泌，与空白对照组相比，动物的存活率可提高至40%～45%。五味子［45］和太子参［46］提取物，均可显著诱导正常细胞生成TNF-α。商路提取物(50 mg/kg)［47］处理小鼠，每周喂食一次，可显著刺激小鼠淋巴细胞增殖，产生IL-2，如果改变喂食频率，一周三次，IL-2 的分泌量则开始下降;此外，当剂量调整为10 mg/kg，每周三次喂食，则可以刺激巨噬细胞分泌IL-1，TNF，推测商路提取物可增强小鼠中巨噬细胞的活性，杀伤病变细胞。蓼大青叶提取物(1～100 mg/kg)［48］可显著增加小鼠脾脏IFN-γ 的分泌。

4 结语

目前，用于临床的抗病毒药物主要有化学药物和疫苗两类，而通过诱生抗病毒细胞因子途径的药物有望成为第三类新型抗病毒药物。药用植物中的天然小分子结构复杂，资源丰富，成药性强，然而目前有关天然小分子诱生抗病毒细胞因子的研究缺乏系统性，尚待进行深入研究。对抗病毒药用植物进行提取分离及纯化，进行体外细胞模型筛选，并开展动物免疫学和动物病毒学研究，进而发现活性天然小分子，通过系统的基础研究，有望研发出诱生机体抗病毒细胞因子的抗感染性疾病的临床药物，我们预计此类相关的研究将成为抗病毒药物研发的新途径。

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