趙京霞 郭玉紅 何莎莎 徐霄龍 崔煦然 劉清泉

(中醫(yī)感染性疾病基礎(chǔ)研究北京市重點(diǎn)實(shí)驗(yàn)室,首都醫(yī)科大學(xué)附屬北京中醫(yī)醫(yī)院,北京市中醫(yī)研究所,北京100010)

NALP3炎性體活化對(duì)膿毒癥發(fā)病過(guò)程中HMGB1釋放的調(diào)節(jié)作用

趙京霞 郭玉紅 何莎莎 徐霄龍 崔煦然 劉清泉

(中醫(yī)感染性疾病基礎(chǔ)研究北京市重點(diǎn)實(shí)驗(yàn)室,首都醫(yī)科大學(xué)附屬北京中醫(yī)醫(yī)院,北京市中醫(yī)研究所,北京100010)

失控性全身炎性反應(yīng)和免疫功能障礙是膿毒癥發(fā)生的主要病理生理過(guò)程。HMGB1是膿毒癥致死效應(yīng)的最重要的晚期炎性遞質(zhì),HMGB1由病原體或早期炎性反應(yīng)分子活化免疫細(xì)胞釋放,在膿毒癥的發(fā)生、發(fā)展過(guò)程中它不僅發(fā)揮促炎效應(yīng),而且還與細(xì)胞免疫功能障礙密切相關(guān)。膿毒癥和內(nèi)毒素血癥中HMGB1的分泌和釋放繼發(fā)于NALP3炎性體的活化。越來(lái)越多的證據(jù)表明,炎性體(尤其是NALP3炎性體)對(duì)單核/巨噬細(xì)胞釋放HMGB1起著重要的調(diào)節(jié)作用。在炎性反應(yīng)過(guò)程中,免疫細(xì)胞主動(dòng)釋放HMGB1的過(guò)程主要以一種炎性反應(yīng)小體參與的、依賴caspase活化的方式進(jìn)行。通過(guò)調(diào)節(jié)NALP3炎性體活化,抑制HMGB1釋放,調(diào)節(jié)免疫功能紊亂狀態(tài),為膿毒癥等免疫紊亂相關(guān)疾病的治療提供了新的策略。

膿毒癥;高遷移率族蛋白B1;NALP3炎性體

免疫功能紊亂在膿毒癥發(fā)生、發(fā)展過(guò)程中具有重要作用。機(jī)體在遭受感染、創(chuàng)傷及大手術(shù)等應(yīng)激情況下,多種炎性反應(yīng)遞質(zhì)大量釋放,導(dǎo)致失控性全身炎性反應(yīng)和免疫功能障礙是膿毒癥發(fā)生的主要病理生理過(guò)程[2]。高遷移率族蛋白B1(High Mobility Group Box-1 Protein,HMGB1)是膿毒癥致死效應(yīng)的最重要的晚期炎性反應(yīng)遞質(zhì)[3]。HMGB1由病原體或早期炎性反應(yīng)分子活化免疫細(xì)胞釋放,在膿毒癥的發(fā)生、發(fā)展過(guò)程中它不僅發(fā)揮促炎效應(yīng),而且還與細(xì)胞免疫功能障礙密切相關(guān)[4]。一方面,HMGB1通過(guò)直接或者間接作用激活樹(shù)突狀細(xì)胞、單核/巨噬細(xì)胞、內(nèi)皮細(xì)胞等,產(chǎn)生更多的炎性反應(yīng)因子,并且與TNF-α、IL-1和MCP-1等分子相互誘生[5],起到放大擴(kuò)散炎性反應(yīng)的作用。另一方面,HMGB1通過(guò)促進(jìn)Treg和DCreg的免疫抑制效應(yīng)[6-7]、引發(fā)機(jī)體免疫耐受、削弱免疫細(xì)胞對(duì)病原體的吞噬清除作用,加重膿毒癥免疫抑制狀態(tài)[8]。并且,HMGB1自身即可誘導(dǎo)膿毒癥的發(fā)生[9]。

膿毒癥患者或動(dòng)物血清及多種組織中均存在HMGB1水平的上升,其水平與臨床嚴(yán)重感染、休克患者及膿毒癥動(dòng)物的病情嚴(yán)重程度及預(yù)后密切相關(guān),針對(duì)HMGB1進(jìn)行干預(yù)則可有效降低膿毒癥動(dòng)物的晚期死亡率[10-12]。由于HMGB1多在膿毒癥開(kāi)始后16～24 h釋放,具有表達(dá)增高晚、持續(xù)時(shí)間長(zhǎng)的特點(diǎn),為改善膿毒癥預(yù)后提供了獨(dú)特的時(shí)間窗,HMGB1成為膿毒癥治療的理想靶標(biāo)[13]。HMGB1主要由激活的單核/巨噬細(xì)胞分泌和釋放。本文重點(diǎn)介紹NALP3炎性體活化對(duì)膿毒癥發(fā)病過(guò)程中HMGB1釋放的調(diào)節(jié)作用。

1 NALP3炎性體及其活化

炎性體(Inflammasome)是一種多蛋白復(fù)合體,可識(shí)別和感受多種微生物產(chǎn)物及細(xì)胞內(nèi)自身代謝性應(yīng)激,各種信號(hào)的刺激均能活化胱天蛋白酶(Cysteinylaspartate-specific Protease,caspase),誘導(dǎo)白介素(interleukin,IL)-1β、IL-18、IL-33等促炎性反應(yīng)細(xì)胞因子的分泌及多種炎性反應(yīng)遞質(zhì)的釋放[14]。NALP3炎性體是NAL炎性體家族成員之一,由核心蛋白NALP3、ASC、Cardinal蛋白和pro-caspase-1構(gòu)成。其核心蛋白NALP3在受到外源性和內(nèi)源性危險(xiǎn)信號(hào)刺激后,通過(guò)招募ASC和pro-caspase-1,完成NALP3炎性體復(fù)合物的裝配和寡聚化[15]。NALP3寡聚體化使pro-caspase-1實(shí)現(xiàn)空間距離的接近,進(jìn)而自身切割形成成熟的caspase-1[16],caspase-1負(fù)責(zé)將無(wú)活性IL-1β前體剪切為成熟IL-1β,并且caspase-1也能水解pro-IL-18和pro-IL-33等前炎性反應(yīng)細(xì)胞因子,使其成為有活性的IL-18和IL-33,從而引起炎性反應(yīng),介導(dǎo)多種感染性疾病的病生理過(guò)程[17]。

2 NALP3炎性體的活化對(duì)HMGB1的釋放的調(diào)節(jié)作用

多項(xiàng)研究表明,在膿毒癥發(fā)病過(guò)程中,存在炎性體(尤其是NALP3炎性體)的異常活化伴隨其底物IL-1β、IL-18的顯著升高[18-19]。炎性體是caspase活化所必需的反應(yīng)平臺(tái)。研究表明,抑制膿毒癥或內(nèi)毒素血癥動(dòng)物caspase活性可以顯著降低血清HMGB1的水平并提高實(shí)驗(yàn)動(dòng)物的生存率[20]。Lamkanfi[21]也發(fā)現(xiàn)在LPS誘導(dǎo)的內(nèi)毒素血癥動(dòng)物模型中,caspase-1基因缺失小鼠與野生型小鼠相比生存時(shí)間顯著延長(zhǎng)伴隨血清中HMGB1水平降低。體外實(shí)驗(yàn)也證實(shí),caspase非特異性抑制劑可阻斷巨噬細(xì)胞HMGB1的分泌[20]。由此可見(jiàn),在炎性反應(yīng)過(guò)程中,免疫細(xì)胞主動(dòng)釋放HMGB1的過(guò)程主要以一種炎性反應(yīng)小體參與的、依賴caspase活化的方式進(jìn)行。

Willingham研究團(tuán)隊(duì)發(fā)現(xiàn)[22],在Nalp3-/-基因缺失小鼠的巨噬細(xì)胞中,肺炎克雷佰氏桿菌誘導(dǎo)小鼠巨噬細(xì)胞釋放HMGB1的作用完全被抑制。另有研究表明[21],野生型小鼠巨噬細(xì)胞在LPS+ATP或LPS+nigericin刺激下,HMGB1分泌水平顯著升高,約為基礎(chǔ)水平的10～15倍。而來(lái)源于Nalp3-/-、ASC-/-、caspase-1-/-3種基因缺失小鼠的巨噬細(xì)胞在受到相應(yīng)刺激時(shí),HMGB1分泌水平未見(jiàn)明顯波動(dòng),與基礎(chǔ)值相當(dāng)。NALP3、ASC、caspase-1是NALP3炎性體的主要成分,以上研究提示炎性體(尤其是NALP3炎性體)的裝配和活化是LPS誘導(dǎo)巨噬細(xì)胞分泌HMGB1的主要調(diào)節(jié)因素。

NALP3炎性體能被一系列不同的內(nèi)源性和外源性因素所刺激。Lucattelli M[23]等發(fā)現(xiàn),選擇性P2X7受體拮抗劑或P2X7受體基因敲除小鼠均可抑制caspase-1活性,減少IL-1β和IL-18釋放,提示P2X7受體參與炎性體的形成及活化。新近研究表明,PKR(Protein kinase R)在P2X7受體途徑介導(dǎo)的炎性體活化過(guò)程中具有重要調(diào)節(jié)作用。Lu等[24]采用不同方法使PKR失活后,LPS誘導(dǎo)巨噬細(xì)胞NALP3炎性體活化及HMGB1釋放的作用被大大削弱,而過(guò)表達(dá)PKR可顯著提高重組NALP3炎性體的活性。并且PKR可以直接和NALP3炎性體中的NALP3蛋白相互作用,從而調(diào)節(jié)炎性體的活化[25-26]。目前多數(shù)研究認(rèn)為[27-28],當(dāng)機(jī)體受到感染后,病原體會(huì)刺激機(jī)體產(chǎn)生過(guò)量的ATP,激活Panx1通道并流出,后者持續(xù)性刺激單核/巨噬細(xì)胞上的P2X7受體,使其聚集活化進(jìn)而開(kāi)放,ATP、PAMP或DAMP通過(guò)該通道進(jìn)入胞質(zhì)促進(jìn)PKR的活化和自磷酸化,活化的PKR與NALP3等炎性體相互作用,激活NALP3炎性體,從而誘導(dǎo)HMGB1的釋放。

3 NALP3炎性體活化/HMGB1釋放與膿毒癥治療

多項(xiàng)動(dòng)物模型研究證實(shí),拮抗HMGB1的作用或抑制HMGB1釋放可顯著提高膿毒癥動(dòng)物的生存率,尤其是多種中藥成分在調(diào)節(jié)HMGB1釋放方面表現(xiàn)出良好的效果,如當(dāng)歸、丹參等[3]。Glyburide是第一個(gè)被發(fā)現(xiàn)具有抑制NALP3炎性體活化作用的小分子抑制劑,對(duì)由LPS+ATP引起的NALP3炎性體活化具有特異性的抑制作用,并可以降低內(nèi)毒素休克引起的動(dòng)物死亡率,其機(jī)制與調(diào)節(jié)P2X7受體途徑有關(guān)[29-30]。Li等[28]報(bào)道甘草主要成分的衍生物carbenoxolone通過(guò)阻斷PKR磷酸化,從而抑制LPS誘導(dǎo)巨噬細(xì)胞NALP3炎性體活化及HMGB1釋放的作用。以上研究提示,調(diào)節(jié)NALP3炎性體活化、抑制HMGB1釋放將為膿毒癥治療的提供更廣闊的前景。

4 結(jié)語(yǔ)

綜上所述,炎性體(尤其是NALP3炎性體)對(duì)單核/巨噬細(xì)胞主動(dòng)釋放HMGB1起著重要的調(diào)節(jié)作用。調(diào)節(jié)NALP3炎性體活化,抑制HMGB1釋放,為膿毒癥治療提供了新的策略。新近研究表明,在膿毒癥過(guò)程中,NALP3炎性體活化后可通過(guò)引起細(xì)胞焦亡(pyroptosis)的形式主動(dòng)釋放HMGB1,但其具體機(jī)制尚未闡明,有待進(jìn)一步的深入。

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(2017-03-17收稿 責(zé)任編輯：王明)

The regulating effect of NALP3 inflammasome on HMGB1 release in the pathogenetic progess of sepsis

Zhao Jingxia,Guo Yuhong,He Shasha,Xu Xiaolong,Cui Xuran,Liu Qingquan

(BeijingKeyLaboratoryofbasicresearchwithTraditionalChineseMedicineoninfectiousdiseases,BeijingHospitalofTraditionalChineseMedicine,affiliatedtoCapitalMedicalUniversity,BeijingInstituteofTraditionalChineseMedicine,Beijing100010,China)

Uncontrolled systemic inflammatory response and immune dysfunction are the major pathophysiological process of sepsis.High mobility group protein B1(HMGB1) is one of the most important terminal inflammatory mediators leading to the lethal effect of severe sepsis.HMGB1 is released by pathogens or immune cells activated by early inflammatory factors,and it not only plays a pro-inflammatory effect in the occurrence and development of sepsis,but also closely related to cellular immune dysfunction.The secretion and release of HMGB1 in sepsis as well as endotoxemia are secondary to the activation of inflammasome.More and more evidences show that inflammasome(especially NALP3) plays an important role regulating the release of HMGB1 for monocytes/macrophages.In the inflammation,the immune cells release HMGB1 actively.This progress require a kind of inflammasomes,and it depends on the activation of caspase activation.It provides a new strategy for the treatment of immune disorders such as sepsis by adjusting the activation of NALP3,restrain the release of HMGB1 and regulating immune dysfunction.

Sepsis;HMGB1;NALP3 inflammasome

國(guó)家自然科學(xué)基金面上項(xiàng)目(編號(hào):81673934);北京市“215”高層次衛(wèi)生技術(shù)人才培養(yǎng)計(jì)劃(編號(hào):2015-3-116);北京市醫(yī)院管理局重點(diǎn)醫(yī)學(xué)專業(yè)發(fā)展計(jì)劃專項(xiàng)(編號(hào):ZYLX201611)

劉清泉(1965.11—)，男，大學(xué)本科，主任醫(yī)師/教授，首都醫(yī)科大學(xué)附屬北京中醫(yī)醫(yī)院院長(zhǎng)，研究方向：中西醫(yī)結(jié)合診治危重病，E-mail:liuqingquan2003@126.com

R278

A

10.3969/j.issn.1673-7202.2017.04.003