胡小鵬，金 哲，賀震旦

深圳大學(xué)醫(yī)學(xué)部醫(yī)學(xué)院藥學(xué)系 深圳大學(xué)生物治療研究所 深圳天然小分子創(chuàng)新藥物工程實(shí)驗(yàn)室，深圳 518060

中醫(yī)藥具有五千多年的歷史，國(guó)內(nèi)外科學(xué)家對(duì)中藥、天然藥、民間藥和民族藥物進(jìn)行了大量的研究，具有抗病毒藥用植物約100 余種［1］，如金銀花(Lonicera japonica)、黃芩(Scutellaria baicalensis)、黃芪(Astragalus membranaceus)、五味子(Schisandra chinensis(Turcz.))、夏枯草(Prunella vulgaris)、連翹(Forsythia suspensa)等(見(jiàn)表1)，據(jù)迄今為止的研究表明天然小分子(Natural micromolecule)為這些抗病毒藥用植物活性的物質(zhì)基礎(chǔ)。

抗病毒細(xì)胞因子(Antiviral cytokines)曾在Nasu K［2］和Fakruddin JM［3］等人發(fā)表的學(xué)術(shù)論文中提及過(guò)，但未曾對(duì)其作出具體的定義。為系統(tǒng)地開(kāi)展細(xì)胞因子抗病毒作用及其作用機(jī)制研究的研究，有必要對(duì)抗病毒細(xì)胞因子進(jìn)行明確地定義:抗病毒細(xì)胞因子是由機(jī)體細(xì)胞分泌的，具有聯(lián)動(dòng)機(jī)體固有免疫和特異性免疫應(yīng)答，進(jìn)而抑制或捕殺體內(nèi)病毒的小分子蛋白?？共《炯?xì)胞因子可分兩大類(lèi):一類(lèi)是可直接作用體細(xì)胞產(chǎn)生抗病毒蛋白，即1)干擾素(Interferon):IFN-α、IFN-β、IFN-γ 和IFN-λ(IL-28、IL-29)［4-7］;2)腫 瘤 壞 死 因 子(Tumor Necrosis Factor)［8］:TNF-α 和TNF-β;干擾素和腫瘤壞死因子可直接激活細(xì)胞內(nèi)JAK(Janus activated kinase)-STAT(signal transducer and activator of transcription)信號(hào)通路［9-13］，隨后引起下游信號(hào)級(jí)聯(lián)反應(yīng)，包括MTOR信號(hào)通路、PI3K 信號(hào)通路、MAPK 信號(hào)通路均被激活，最終啟動(dòng)細(xì)胞核中一些關(guān)鍵的蛋白基因進(jìn)行轉(zhuǎn)錄，翻譯出抗病毒蛋白進(jìn)而對(duì)細(xì)菌、病毒以及癌細(xì)胞等產(chǎn)生防御作用［14］(見(jiàn)圖1)。另一類(lèi)是能激活巨噬細(xì)胞、自然殺傷細(xì)胞(NK 細(xì)胞)和毒性T 淋巴細(xì)胞(CTL)等細(xì)胞，進(jìn)而捕殺或吞噬體內(nèi)病毒的細(xì)胞因子(見(jiàn)圖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］。

基于細(xì)胞因子對(duì)捕殺或抑制病毒方面的作用，本文綜述了國(guó)內(nèi)外迄今為止報(bào)道的可誘生抗病毒細(xì)胞因子的天然小分子(見(jiàn)圖2，圖3)，其中以多酚類(lèi)、苷類(lèi)居多，還有部分物質(zhì)基礎(chǔ)未明確的提取物，有待進(jìn)一步研究。

1 多酚類(lèi)化合物(Polyphenolic compounds)

白藜蘆醇(1，Resveratrol，10 mol/L)能上調(diào)人外周血單核細(xì)胞IL-1β、IL-6、TNF-α，調(diào)節(jié)免疫細(xì)胞功能［21］。從姜黃(Curcuma longa)根莖分離的天然多酚姜黃素(2，Curcumin)，通過(guò)激活人細(xì)胞內(nèi)MAPK以及MTOR 等信號(hào)通路，調(diào)節(jié)下游STATs 蛋白磷酸化水平，控制TNF-α、IL-1 和IL-6 的表達(dá)分泌［22］，姜黃素還可以顯著的調(diào)控TRAIL(TNF 相關(guān)凋亡誘導(dǎo)配基)，有研究顯示姜黃素(10 μmol/L 和25 μmol/L)和TRAIL(20 ng/mL)聯(lián)合使用能夠促進(jìn)胞內(nèi)半胱天冬酶的前體Procaspase-3，8，9 的分裂，并使得線粒體釋放細(xì)胞色素C，導(dǎo)致細(xì)胞毒性的增強(qiáng)［23］。茶多酚復(fù)方制劑(50～150 mg/kg)可增強(qiáng)小鼠淋巴細(xì)胞增殖能力，提高血清溶菌酶活性，上調(diào)血清中IFN-γ 分泌［24］。番茄(Lycopersicum esculentum)的多酚提取物能夠調(diào)節(jié)牛中性粒細(xì)胞TNF-α 基因的表達(dá)，加熱處理的提取物可顯著上調(diào)IFN-γ 的分泌［25］，具有增強(qiáng)動(dòng)物和人類(lèi)固有免疫的潛在活性。由咖啡酸(3，Caffeic acid)、阿魏酸(4，F(xiàn)erulic acid)和對(duì)香豆酸(5，p-Coumaric acid)酶促聚合的多酚(20～100 μg/mL)，通過(guò)和T 細(xì)胞表面CD4+蛋白結(jié)合，激活下游信號(hào)通路后上調(diào)小鼠脾臟細(xì)胞中的IFN-γ 和GM-CSF(Granulocyte-macrophage Colonystimulating Factor)分泌量［26］，增強(qiáng)機(jī)體固有免疫。

從Cocoa 提取的黃酮類(lèi)化合物，能夠減少小鼠腹膜巨噬細(xì)胞活性氧和NO 的生成，上調(diào)IFN-γ 和TNF-α 的作用［27］。中藥黃芪(Astragalus membranaceus)和黃芩(Scutellaria baicalensis)是傳統(tǒng)的抗病毒藥材，主含槲皮素(6，Quercetin)、黃芩素(7，Baicalein)等天然小分子。Nair［28］等人報(bào)道，槲皮素(5 μmol/L)可調(diào)節(jié)外周血單核細(xì)胞IFN-γ 和IL-4 的分泌，從而促使Th0 細(xì)胞向Th1 細(xì)胞分化;隨后進(jìn)行了攻毒試驗(yàn)，發(fā)現(xiàn)誘生的IFN-γ 具有很強(qiáng)的抗VSV(vesicular stomatitis virus)活性。Johari 等［29］報(bào)道槲皮素和黃芩素抑制JEV(Japanese Encephalitis Virus)的IC50分別為212.1、14.28 μg/mL，提示黃芩素比槲皮素具有更好的抗JEV 作用。有研究表明，黃芩素可直接抑制病毒血凝素和神經(jīng)氨酸酶［30］。

桂皮醛(13，Cinnamaldehyde，1 mg/kg)［31，32］可上調(diào)小鼠血清中IL-2、IL-10 的分泌量，其誘生機(jī)制可能與Toll 樣受體的激活有關(guān)。從百屈菜中提取得到 血 根 堿(14，6-acetonyl-5，6-dihydrosanguinarine)［33］，體外實(shí)驗(yàn)證實(shí)其可誘導(dǎo)巨噬細(xì)胞和樹(shù)突狀細(xì)胞分泌TFN-α、IL-6、IL-8 等抗病毒細(xì)胞因子，對(duì)其誘生機(jī)制的研究表明，血根堿可激活MAPK 通路中ERK，NF-kB 循環(huán)活化機(jī)制也參與其中，共同調(diào)控了下游抗病毒細(xì)胞因子的表達(dá)。朱曉新等人［34］從紫萁貫眾分離得到四種多酚化合物: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)可激活胞內(nèi)NF-kB 通路，可顯著上調(diào)小鼠腹膜細(xì)胞分泌TNF、IL-1β、IFN-γ 等，誘生機(jī)制可能和iNOS(一氧化氮合成酶)的激活和NO 的生成有關(guān)。

2 苷類(lèi)化合物(Glycosides)

研究表明苷類(lèi)化合物具有抗腫瘤抗病毒活性［35］;桔梗(Platycodon grandiflorum)中分離得到的單體桔梗苷D(8，Platycodin D，0.0016～0.2 μg/mL)［36］，能夠上調(diào)IL-2、IFN-γ 的mRNA 表達(dá)水平，調(diào)控Th1/Th2 細(xì)胞的轉(zhuǎn)錄因子T-bet 和CATA-3 表達(dá)，同時(shí)，桔梗苷D 可以提高OVA 致敏小鼠抗體水平，具有免疫調(diào)節(jié)作用。王先遠(yuǎn)等［37］研究苦瓜總皂苷對(duì)衰老小鼠免疫功能的影響，發(fā)現(xiàn)苦瓜總皂苷不僅可上調(diào)血清中IL-2 水平，而且促進(jìn)脾臟分泌IL-2及腹腔巨噬細(xì)胞分泌TNF-α，其機(jī)制可能與DNA 多聚酶II 活性增強(qiáng)有關(guān);苦瓜總皂苷促進(jìn)IL-2 分泌，改變T 細(xì)胞亞群的組成，使機(jī)體免疫狀態(tài)趨向年輕化。徐先祥等［38］人報(bào)道甘草皂苷(9，0.5 mg/mL)可抑制98%以上的HIV 增殖，0.125 mg/mL 時(shí)，可抑制98%以上的HIV 的增殖，并可抑制50%斑塊的形成，其作用機(jī)制是誘生干擾素和增強(qiáng)NK 細(xì)胞的活性抵抗HIV。柚皮苷(17，naringin，20，40 and 80 mg/kg)［39］可上調(diào)Th2 型細(xì)胞分泌IL-4、IL-6、IL-10等。

表1 抗病毒藥用植物Table 1 Antivial Medical Plants

注:* 已報(bào)道的可誘生抗病毒細(xì)胞因子的天然小分子Note:* These natural micromolecules have been reported to induce antiviral cytokines.

圖1 天然小分子誘生細(xì)胞因子抗病毒作用機(jī)制Fig.1 The mechanisms of antiviral activity of antiviral cytokines induced by natrual micromolecule

圖2 誘生抗病毒細(xì)胞因子的天然小分子1～12 的化學(xué)結(jié)構(gòu)式Fig.2 The chemical structure of natural micromolecule 1-12 with the function of inducing antiviral cytokines

圖3 誘生抗病毒細(xì)胞因子的天然小分子13～19 的化學(xué)結(jié)構(gòu)式Fig.3 The chemical structure of natural micromolecule 13-19 with the function of inducing antiviral cytokines

另外，本課題組開(kāi)展天然小分子誘生機(jī)體抗病毒細(xì)胞因子的研究中，發(fā)現(xiàn)天麻素(10，Gastrodin，0～1000 μmol/L)、迎春花素(11，Jasminin，1-100 μmol/L)能夠顯著上調(diào)RAW246.7 細(xì)胞分泌TNFα;從苦丁茶紫莖女貞(Ligustrum purpurascens)葉提取得到苯丙素總苷(crude phenylethanoid glycosides)，屬多酚類(lèi)化合物，活性測(cè)試表明，苯丙素總苷(0.22～1.32 g/kg)具有免疫增強(qiáng)作用［49］;進(jìn)一步研究還發(fā)現(xiàn)，從苯丙素總苷分離得到的單體阿克苷(12，acteoside，0.8～200 μg/mL)，能夠顯著上調(diào)Balb/c 小鼠體內(nèi)IFN-γ 的表達(dá)水平，動(dòng)物攻毒實(shí)驗(yàn)證實(shí)可顯著抑制小鼠體內(nèi)病毒載量。

3 其他

大蒜辣素(18，Allitridin，25 mg/kg/day)［40］可調(diào)節(jié)CMV 感染小鼠體內(nèi)Foxp3 蛋白表達(dá)，上調(diào)機(jī)體IFN-γ 和IL-4 分泌，恢復(fù)CMV 攻擊造成的免疫系統(tǒng)紊亂。青蒿素(19，Artemisinin)［41］可顯著刺激巨噬細(xì)胞分泌IL-12，其誘生機(jī)制和JNK 磷酸化的抑制有關(guān)。

滑菇(Pholiota nameko)提取物(20～60 mg/kg)［42］能夠上調(diào)白介素、腫瘤壞死因子、干擾素等多種抗病毒細(xì)胞因子的分泌。

此外，許多中藥提取物具有免疫增強(qiáng)作用［43］，如黃芪提取物(100～200 μg/mL)、板藍(lán)根提取物(100～200 μg/mL)能夠刺激雞外周血淋巴細(xì)胞IFN-γ 的mRNA 的表達(dá)量，并對(duì)NDV(Newcastle disease virus)感染有抑制作用。人參葉提取物［44］可顯著上調(diào)感染H5N1 小鼠和雪貂肺部IFN-γ 分泌，與空白對(duì)照組相比，動(dòng)物的存活率可提高至40%～45%。五味子［45］和太子參［46］提取物，均可顯著誘導(dǎo)正常細(xì)胞生成TNF-α。商路提取物(50 mg/kg)［47］處理小鼠，每周喂食一次，可顯著刺激小鼠淋巴細(xì)胞增殖，產(chǎn)生IL-2，如果改變喂食頻率，一周三次，IL-2 的分泌量則開(kāi)始下降;此外，當(dāng)劑量調(diào)整為10 mg/kg，每周三次喂食，則可以刺激巨噬細(xì)胞分泌IL-1，TNF，推測(cè)商路提取物可增強(qiáng)小鼠中巨噬細(xì)胞的活性，殺傷病變細(xì)胞。蓼大青葉提取物(1～100 mg/kg)［48］可顯著增加小鼠脾臟IFN-γ 的分泌。

4 結(jié)語(yǔ)

目前，用于臨床的抗病毒藥物主要有化學(xué)藥物和疫苗兩類(lèi)，而通過(guò)誘生抗病毒細(xì)胞因子途徑的藥物有望成為第三類(lèi)新型抗病毒藥物。藥用植物中的天然小分子結(jié)構(gòu)復(fù)雜，資源豐富，成藥性強(qiáng)，然而目前有關(guān)天然小分子誘生抗病毒細(xì)胞因子的研究缺乏系統(tǒng)性，尚待進(jìn)行深入研究。對(duì)抗病毒藥用植物進(jìn)行提取分離及純化，進(jìn)行體外細(xì)胞模型篩選，并開(kāi)展動(dòng)物免疫學(xué)和動(dòng)物病毒學(xué)研究，進(jìn)而發(fā)現(xiàn)活性天然小分子，通過(guò)系統(tǒng)的基礎(chǔ)研究，有望研發(fā)出誘生機(jī)體抗病毒細(xì)胞因子的抗感染性疾病的臨床藥物，我們預(yù)計(jì)此類(lèi)相關(guān)的研究將成為抗病毒藥物研發(fā)的新途徑。

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