楊顏銥 陳 蕓 高 爽 劉 旗 陳 憧 姚淑華 鄧俊良

(四川農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院，動物疫病與人類健康四川省重點實驗室，環(huán)境公害與動物疾病四川省高校重點實驗室，成都611130)

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抗菌肽抑制脂多糖誘導(dǎo)的炎癥反應(yīng)

楊顏銥 陳 蕓 高 爽 劉 旗 陳 憧 姚淑華 鄧俊良*

(四川農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院，動物疫病與人類健康四川省重點實驗室，環(huán)境公害與動物疾病四川省高校重點實驗室，成都611130)

抗菌肽是一種對多重耐藥菌株表現(xiàn)出特異性抗菌機(jī)制的小分子多肽。同時，抗菌肽還具有抗炎活性，可以通過直接中和脂多糖(LPS)、抑制生物性炎癥因子的產(chǎn)生來減輕炎癥反應(yīng)；亦可通過趨化白細(xì)胞、促進(jìn)免疫細(xì)胞增殖等來影響獲得性免疫，從而調(diào)節(jié)宿主免疫系統(tǒng)發(fā)揮保護(hù)作用。本文綜述了近年來LPS誘導(dǎo)炎癥產(chǎn)生的機(jī)制及抗菌肽抑制LPS誘導(dǎo)炎癥反應(yīng)的作用機(jī)理。

抗菌肽；脂多糖；炎癥因子；抑制炎癥機(jī)理

脂多糖(lipopolysaccharide，LPS)也被稱為內(nèi)毒素，是革蘭氏陰性菌外膜的主要成分，被認(rèn)為是與革蘭氏陰性菌感染相關(guān)的內(nèi)毒素休克發(fā)病機(jī)制中的一個關(guān)鍵分子[1]，多在細(xì)胞分裂、死亡或抗生素治療細(xì)菌感染的過程中釋放出[2]。LPS對細(xì)菌生存而言必不可少，它通過建立一個有效的滲透性屏障來阻止各種抗菌化合物入侵，包括疏水性抗生素、抗菌肽等[3]?？咕氖且环N具有生物活性的帶正電荷及具備疏水性和兩親性的小分子多肽。研究證實天然的陽離子抗菌肽可以防止細(xì)菌、病毒和寄生蟲等多種感染[4-5]，還能直接中和LPS，抑制炎性細(xì)胞因子的過度產(chǎn)生和釋放從而控制炎癥反應(yīng)，并通過免疫調(diào)節(jié)減少炎性損傷，在炎癥過程中具有重要的作用[6-7]。近段時間以來，抗菌肽因其抗感染性能可作為治療人類以及動植物疾病的替代藥物而備受關(guān)注[8]。本文綜述了近年來LPS誘導(dǎo)炎癥產(chǎn)生的機(jī)制及抗菌肽抑制LPS誘導(dǎo)炎癥反應(yīng)的作用機(jī)理。

1 LPS誘導(dǎo)炎癥反應(yīng)

1.1 LPS的化學(xué)結(jié)構(gòu)

LPS是革蘭氏陰性桿菌細(xì)胞壁結(jié)構(gòu)與功能的重要組成部分，由O-特異性多糖鏈、核心寡聚糖及類脂A構(gòu)成，是帶有負(fù)電荷磷酸基團(tuán)的疏水性長鏈脂肪酸(圖1)[9]。其中類脂A是LPS結(jié)構(gòu)中最保守的部分，在LPS的生物活性中起主要調(diào)控作用[10]。LPS的核心寡聚糖和磷酸基團(tuán)都帶有負(fù)電荷，提示LPS對陽性離子具有極高的親和性[1]。

1.2 LPS誘導(dǎo)炎癥反應(yīng)的信號通路

免疫細(xì)胞可識別病原體相關(guān)分子模式(pathogen-associated molecular patterns,PAMPs)，在炎癥反應(yīng)中起重要作用[11]。感染時，PAMPs之一的LPS與免疫細(xì)胞表面最主要的受體之一——CD14結(jié)合并通過脂多糖結(jié)合蛋白(lipopolysaccharide binding protein，LBP)形成LPS-LBP-CD14三聯(lián)復(fù)合物作用于Toll樣受體4(Toll-like receptors 4，TLR4)從而啟動跨膜信號轉(zhuǎn)導(dǎo)，發(fā)揮致炎效應(yīng)[12]。LBP是一種介導(dǎo)LPS與靶細(xì)胞表面受體CD14結(jié)合的關(guān)鍵載體蛋白。LBP的N端是LBP與LPS的結(jié)合位點，尤其是富含疏水性氨基酸和堿性氨基酸的LBP109～133位殘基容易與LPS結(jié)合；而C端則是其與CD14的結(jié)合位點。Toll樣受體(Toll-like receptors，TLR)是第1個被發(fā)現(xiàn)并明確特征的模式識別受體(pattern recognition receptor,PRR)，TLR4已被確認(rèn)為可識別LPS的受體[13]。

Polysaccharide：多糖；Glycophospholipid：磷脂；Core：核心寡聚糖；Outer core：外核心寡聚糖；Inner core：內(nèi)核心寡聚糖；O-specific chain：O-特異性多糖鏈；Lipid A：類脂A；Hep：L-甘油基-D-甘露庚糖 L-glycero-D-manno-heptose；Kdo：2-酮基-3-脫氧辛酸 2-keto-3-deoxyoctulosonic acid；GlcN：氨基葡萄糖 glucosamine；波浪線為脂肪酸 Zig-zag lines mean fatty acids。

LPS參與細(xì)胞介導(dǎo)的炎癥反應(yīng)時有2條不同的信號轉(zhuǎn)導(dǎo)通路。第1條是髓樣分化因子88(myeloid differentiation factor 88,MyD88)依賴途徑，第2條是含有Toll/(白細(xì)胞介素-1)IL-1受體(TIR)結(jié)構(gòu)域誘導(dǎo)干擾素β(TRIF)依賴途徑，兩者都由LPS和TLR4的關(guān)聯(lián)觸發(fā)[14]。盛金良等[15]的試驗結(jié)果表明，LPS能夠很快誘導(dǎo)TLR4的表達(dá)水平升高，在LPS作用20 min后即達(dá)到較高水平，且在整個試驗過程中保持較高水平。

LPS/TLR4信號通道的激活受到多種信號蛋白或受體的調(diào)控(圖2)[16]。髓樣分化蛋白-2(myeloid differentiation protein-2，MD-2)是一種特殊的外分泌蛋白，能幫助TLR4識別LPS。首先，MD-2與TLR4在免疫細(xì)胞表面形成TLR4-MD-2復(fù)合體，當(dāng)LPS進(jìn)入血液循環(huán)時，LBP作為載體蛋白將LPS傳遞給CD14，后者進(jìn)一步凝集LPS并將其呈遞給TLR4-MD-2復(fù)合體。最后，MD-2識別并與LPS結(jié)合形成TLR4-MD-2-LPS三聚體，激活TLR4跨膜信號通路。MyD88依賴信號通路的激活導(dǎo)致絲裂原活化蛋白激酶(mitogen-activated protein kinases，MAPKs)、抑制性κB激酶(inhibitor of nuclear factor kappa-B kinase，IKK)和核轉(zhuǎn)錄因子-κB(nuclear factor-κB，NF-κB)磷酸化，最終導(dǎo)致促炎細(xì)胞因子如腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素-1β(IL-1β)、白細(xì)胞介素-6(IL-6)、誘導(dǎo)型一氧化氮合酶(iNOS)、環(huán)氧合酶-2(COX-2)等的表達(dá)[17]。MAPKs通路主要包括p38、c-Jun氨基末端激酶(c-Jun N-terminal kinase，JNK)和細(xì)胞外信號調(diào)節(jié)激酶(extracellular signal-regulated kinase，ERK)[18]。許多研究表明，抑制p38、JNK和ERK 3條通路的磷酸化可達(dá)到抗炎目的。p38的激活是促炎細(xì)胞因子表達(dá)的先決條件，抑制p38通路已被證實可在人體內(nèi)毒素血癥中發(fā)揮抗炎作用[19]。

2 抗菌肽免疫調(diào)節(jié)功能的分子機(jī)制

當(dāng)PAMPs與PRR相互作用時，免疫細(xì)胞分泌趨化因子及防御素α和LL-37等抗菌肽[20]?？咕目赏ㄟ^誘導(dǎo)肥大細(xì)胞發(fā)生脫顆粒作用，釋放組胺和前列腺素D2，引起血管舒張并導(dǎo)致血液中免疫細(xì)胞的釋放，進(jìn)而誘導(dǎo)巨噬細(xì)胞凋亡和淋巴細(xì)胞活化來調(diào)節(jié)宿主免疫力。此外，抗菌肽還可以增強(qiáng)成纖維細(xì)胞的趨化性，促進(jìn)內(nèi)皮細(xì)胞和淋巴細(xì)胞的增殖，加速傷口愈合。例如，人抗菌肽LL-37可與甲酰肽受體1(FPR1)相互作用使單核細(xì)胞、中性粒細(xì)胞、淋巴細(xì)胞和T淋巴細(xì)胞趨化[21]。研究表明經(jīng)過活化的FPR1和LL-37不僅對白細(xì)胞有趨化作用，還能增強(qiáng)白細(xì)胞的黏附力、吞噬能力，促進(jìn)氧中間產(chǎn)物的釋放，增強(qiáng)對細(xì)菌的殺傷力，從而增強(qiáng)免疫力[22]。

LPS：脂多糖 lipopolysaccharide；LBP：脂多糖結(jié)合蛋白 lipopolysaccharide binding protein；MD-2：髓樣分化蛋白-2 myeloid differentiation protein-2；TLR：Toll樣受體 Toll-like receptor；Cytoplasm：細(xì)胞漿；ERK1/2：細(xì)胞外調(diào)節(jié)蛋白激酶 extracellular regulated protein kinases；JNK：c-Jun氨基末端激酶 c-Jun N-terminal kinase；MyD88：髓樣分化因子88 myeloid differentiation factor 88；IRAK：白細(xì)胞介素-1受體相關(guān)激酶 interleukin-1 receptor-associated kinase；TRAF6：腫瘤壞死相關(guān)因子6 tumor necrosis factor receptor-associated factor 6；SRE：血清反應(yīng)組件 serum response element；AP-1：激活子蛋白-1 activator protein-1；NF-κB：核轉(zhuǎn)錄因子-κB nuclear factor-κB；CRE；cAMP反應(yīng)組件 cAMP response element；TNF-α：腫瘤壞死因子-α tumor necrosis factor-α；IL-6：白細(xì)胞介素-6 interleukin-6；IL-8：白細(xì)胞介素-8 interleukin-8。

3 抗菌肽與LPS相互作用時結(jié)構(gòu)與活性的關(guān)系

針對抗菌肽的結(jié)構(gòu)與活性關(guān)系的詳細(xì)研究表明，有許多抗菌肽都具備中和LPS的活性，但是不同結(jié)構(gòu)的肽中和LPS的活性存在著顯著的差異。Heinbockel等[23]研究了Hbγ-35和Pep19-2.5 2種抗菌肽對LPS聚合構(gòu)造的影響。Hbγ-35作用時LPS被分解為立方體結(jié)構(gòu)，且Hbγ-35增加了LPS誘導(dǎo)的人體單核細(xì)胞分泌TNF-α。與之相反的是，Pep19-2.5使LPS從立方體結(jié)構(gòu)轉(zhuǎn)變成多層結(jié)構(gòu)，從而抑制了TNF-α分泌。

Kaconis等[24]研究了一系列合成肽對LPS的中和作用，表明LPS形成多層結(jié)構(gòu)的能力與抑制LPS刺激產(chǎn)生細(xì)胞因子直接相關(guān)，且合成肽與LPS的結(jié)合在抗菌肽的抗菌活性和抗炎活性均起著重要的作用。rBPI21是中性粒細(xì)胞N末端的殺菌/通透性增加蛋白(bactericidal/permeability-increasing protein，BPI)片段，它能夠選擇性的抑制革蘭氏陰性菌，且對LPS有很強(qiáng)的親和力。rBPI21在與LPS相互作用時可引起含有豐富的磷脂酰膽堿的革蘭陰性細(xì)菌膜泄漏[25]。

事實上，許多在結(jié)構(gòu)上不夠優(yōu)化的天然抗菌肽，可以通過適當(dāng)?shù)剡M(jìn)行氨基酸取代改善其活性[26]。例如Nan等[27]將抗菌肽LL-37的類似物a4中5、15位的苯丙氨酸替換為色氨酸后顯著提高了它中和LPS的活性。

Singh等[28]通過一系列來源于S1肽酶的抗菌肽來探究靜電作用在抗菌肽與LPS相互作用時發(fā)揮的作用，證實在很大程度上抗菌肽與磷脂膜結(jié)合是依賴于肽的兩親性構(gòu)象，而與LPS結(jié)合則取決于抗菌肽的凈電荷及疏水性。同樣，Andr?等[29]報道LPS與抗菌肽NK-2結(jié)合時不僅依靠靜電作用，疏水作用也非常關(guān)鍵。Lee等[30]根據(jù)牛源抗菌肽BAMP-27合成了一種含18個氨基酸殘基的肽，BAMP-18以及其類似物(BAMP-18-W、BAMP-18-L、BAMP-18-I和BAMP-18-F)。結(jié)果證實BAMP-18及類似物能明顯抑制TNF-α和一氧化氮(NO)。盡管BMAP-18-W疏水性低于BMAP-18-L，其中和LPS的能力卻更強(qiáng)。說明抗菌肽中和LPS的活性不僅與攜帶的正電荷數(shù)及疏水性有關(guān)，還存在其他的影響因素。

4 抗菌肽直接中和LPS的作用機(jī)制

抗菌肽來源眾多，包括來源于昆蟲的天蠶素與蜂毒素的混合抗菌肽CEMA、人抗菌肽LL-37、牛源抗菌肽BAMP-27、合成的小分子陽離子抗菌肽等，其中大多數(shù)抗菌肽都被證實能顯著抑制LPS致炎癥反應(yīng)，并防止內(nèi)毒素血癥[31]。

Fang等[32]試驗表明，成熟的U937細(xì)胞在受到LPS刺激后會激活NF-κB通路并釋放TNF-α。當(dāng)U937細(xì)胞與LBP、LPS共同處理時，NF-κB活化的程度及TNF-α的表達(dá)量均顯著升高。證實LBP能加劇LPS誘導(dǎo)的炎癥反應(yīng)。而多肽P1和MP12對LPS-LBP復(fù)合物誘導(dǎo)的NF-κB的活化和TNF-α表達(dá)的具有明顯的抑制作用，均較LPS單獨處理時更低。證實多肽P1和MP12可以和CD14與LBP結(jié)合位點結(jié)合，阻止LPS和CD14通過LBP結(jié)合，抑制LPS釋放炎癥因子。

Nagaoka等[33]研究表明，抗菌肽CAP18、CAP11以及LL-37均可抑制LPS與CD14結(jié)合從而抑制LPS誘導(dǎo)巨噬細(xì)胞表達(dá)產(chǎn)生TNF-α。表明抗菌肽不僅可以結(jié)合LPS，也可以和免疫細(xì)胞膜受體CD14結(jié)合從而此抑制LPS誘導(dǎo)炎癥。最近，該團(tuán)隊的研究顯示，LL-37通過抑制LPS受體復(fù)合物的集合以及受體CD14、TLR4和磷酸化的JNK來阻止LPS誘導(dǎo)內(nèi)皮細(xì)胞凋亡[34]。應(yīng)當(dāng)指出的是，LL-37對TNF-α的表達(dá)調(diào)控機(jī)制是有待進(jìn)一步研究的。一方面，LL-37顯示出很強(qiáng)的預(yù)防內(nèi)毒素休克小鼠模型合成TNF-α的能力；另一方面，Babolewska等[35]最近的研究表明LL-37可誘導(dǎo)大鼠結(jié)締組織中成熟的肥大細(xì)胞脫粒與遷移，并刺激這些細(xì)胞合成多種細(xì)胞因子，其中包括TNF-α。

Liu等[36]報道，CLP-19通過與LPS競爭結(jié)合LBP從而抑制LPS誘導(dǎo)炎癥反應(yīng)信號通路，明顯減少細(xì)胞因子TNF-α的釋放。而當(dāng)抗菌肽、LPS和LBP三者同時存在時，抗菌肽能夠成功阻止LPS結(jié)合LBP但很少分解LPS-LBP復(fù)合物。Monisha[37]發(fā)現(xiàn)，預(yù)處理后的抗菌肽MBI-28與吞噬細(xì)胞共同培養(yǎng)1 h，除去上清液后在新的培養(yǎng)基中加入LPS，MBI-28仍然能夠抑制巨噬細(xì)胞表達(dá)產(chǎn)生的TNF-α，提示抗菌肽還存在另外一個機(jī)制抑制LPS誘導(dǎo)的炎癥，具體的作用機(jī)制還需進(jìn)一步的研究。

最近的研究表明LL-37對細(xì)胞凋亡擁有驚人的抑制能力，敗血性休克的另一個特點就是LPS和ATP通過促進(jìn)半胱氨酸特異性蛋白水解酶1(cystein aspartate-specific proteases 1,caspase-1)的過表達(dá)導(dǎo)致細(xì)胞死亡，大量的促炎性細(xì)胞因子包括IL-1β在級聯(lián)反應(yīng)的作用下細(xì)胞外釋放，其中IL-1β是引起全身性休克組織損傷的主要因子。Hu等[38]就證實了LL-37不僅可以抑制LPS結(jié)合CD14和TLR4，還能夠抑制ATP驅(qū)動的P2X7受體激活以及caspase-1的形成。

Swangchan等[39]試驗證明，牛子宮內(nèi)膜細(xì)胞在受到LPS刺激時，舌抗菌肽(LAP)、氣管抗菌肽(TAP)、S100A8、S100A9和S100A12等的mRNA含量會增加，表明發(fā)生炎癥時，抗菌肽的基因表達(dá)將會被上調(diào)。同時也證實了抗菌肽的一些基因(TAP和S100A9等)可作為子宮炎癥的診斷標(biāo)志物。

Kim等[40]在原有的抗菌肽GNU7基礎(chǔ)上加入幾種設(shè)計合理的LPS靶向肽，包括乳鐵蛋白28～34位的氨基酸(Lf28～34)，BPI的84～99位氨基酸(BPI84～99)以及從頭合成肽(Syn)，從而合成了Lf-GNU7、BPI-GNU7、Syn-GNU7 3種合成抗菌肽。試驗結(jié)果證明，與GNU7相比，合成肽的抗菌活性提升了8～32倍，其中SYN-GNU7具有最強(qiáng)的中和LPS及抗炎活性。說明在原有抗菌肽的基礎(chǔ)上加入少量LPS靶向肽可顯著提高抗菌肽的抗炎活性。

抗菌肽抗炎的作用模式比較復(fù)雜，包含多個分子機(jī)制。較為重要的有如下3種：1)通過阻塞LPS脂質(zhì)A的抗原表位來防止LPS與LBP結(jié)合從而避免NF-κB通路激活；2)通過抗菌肽的吸附作用使巨噬細(xì)胞和單核細(xì)胞膜的正電荷積聚，反過來使膜與LPS結(jié)合；3)抗菌肽引發(fā)的吞噬作用，中和電荷以及破壞LPS集合物[41]。

5 小 結(jié)

近年來的研究證實抗菌肽具備良好的抑制炎癥以及治療LPS引起的敗血性休克的潛能。此外，抗菌肽還可以調(diào)節(jié)炎癥相關(guān)信號通路和轉(zhuǎn)錄因子、調(diào)節(jié)免疫活性相關(guān)分子，調(diào)節(jié)機(jī)體免疫功能來實現(xiàn)抗炎作用。雖然抗菌肽的應(yīng)用前景廣闊，但就目前而言大規(guī)模應(yīng)用還暫時不可行，部分陽離子抗菌肽的細(xì)胞毒性問題[42]、抗菌肽的不穩(wěn)定性[43-44]等都亟待解決。隨著對抗菌肽研究的不斷深入，不斷開發(fā)合適的抗菌肽劑型，將為其在炎癥的臨床治療帶來更為廣闊的應(yīng)用前景，有望作為新的抗炎藥物應(yīng)用于各種炎癥性疾病的治療。

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*Corresponding author, professor, E-mail: dengjl213@126.com

(責(zé)任編輯 田艷明)

Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation

YANG Yanyi CHEN Yun GAO Shuang LIU Qi CHEN Chong YAO Shuhua DENG Junliang*

(KeyLaboratoryofAnimalDisease&HumanHealthofSichuanProvince,KeyLaboratoryofEnvironmentalHazardandAnimalDiseaseofSichuanProvince,CollegeofVeterinaryMedicine,SichuanAgriculturalUniversity,Chengdu611130,China)

Antimicrobial peptides (AMPs) are a kind of small molecular polypeptide which show the specific antibacterial mechanism to multiple drug-resistant strains. In addition, with anti-inflammatory activity, AMPs relieve inflammation by directly neutralizing lipopolysaccharide (LPS) and inhibiting the release of biological inflammatory factors. Meanwhile, they also affect the acquired immunity through the chemotaxis of leukocytes and promoting the proliferation of the immune cells to adjust the host immune system and play a protective role. This essay summarized the mechanism of LPS induced inflammation and the AMPs to inhibit LPS induced inflammation in recent years.[ChineseJournalofAnimalNutrition, 2016, 28(12)：3770-3776]

AMPs; LPS; inflammatory factors; mechanism of inhibiting inflammation

10.3969/j.issn.1006-267x.2016.12.008

2016-06-21

教育部“長江學(xué)者和創(chuàng)新團(tuán)隊發(fā)展計劃”創(chuàng)新團(tuán)隊項目(IRTO848)；四川農(nóng)業(yè)大學(xué)雙支計劃(03571537)

楊顏銥(1991—)，女，四川南充人，碩士研究生，從事反芻動物疾病研究。E-mail： ash_lee_love@yeah.net

*通信作者：鄧俊良，教授，博士生導(dǎo)師，E-mail： dengjl213@126.com

S811

A

1006-267X(2016)12-3770-07