吳麗潔,張霽,張丹,楊延婷,楊玲,智方圓,吳凌翔,謝晨,馬曉芃,

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NLRP3炎癥小體與炎癥性腸病的關(guān)系及針灸調(diào)節(jié)作用研究進展

吳麗潔1,張霽1,張丹2,楊延婷1,楊玲2,智方圓1,吳凌翔2,謝晨2,馬曉芃1,2

(1.上海中醫(yī)藥大學,上海 201203;2.上海市針灸經(jīng)絡(luò)研究所,上海 200030)

炎癥性腸病(inflammatory bowel disease, IBD)是一組慢性非特異性腸道炎癥性疾病,包括潰瘍性結(jié)腸炎(UC)和克羅恩病(CD)。炎癥小體作為一種大分子復(fù)合蛋白,在炎癥發(fā)生發(fā)展與機體固有免疫中發(fā)揮了重要作用。其中NLRP3在IBD的發(fā)生發(fā)展與維持腸道穩(wěn)態(tài)的作用機制中起到了雙向調(diào)節(jié)的作用。該文綜述了NLRP3炎性小體在IBD中的研究進展以及針灸對其的調(diào)節(jié)作用及可能機制,以期為針灸治療炎癥性腸病的臨床和機制研究提供新思路。

炎癥性腸病;針灸療法;炎癥小體;NLRP3

炎癥性腸病(inflammatory bowel disease, IBD)是一組影響胃腸道功能的慢性炎癥性疾病,主要包括克羅恩病(CD)和潰瘍性結(jié)腸炎(UC)[1]。其發(fā)病機制尚未完全明確,可能與腸道上皮屏障功能的改變、腸道微生物失調(diào)、腸道先天免疫和飲食等因素相關(guān)[2]。NLRP3作為炎癥小體的其中一個亞型,是目前研究最多也是最具特點的一員,對炎癥性腸病的調(diào)控具有雙向調(diào)節(jié)作用[3]。大量的臨床實踐已證明,針灸治療炎癥性腸病療效確切[4-27],機制研究也逐步深入。探討NLRP3在IBD中的分子調(diào)節(jié)機制以及針灸干預(yù)作用,為針灸治療IBD提供可靠的科學依據(jù)具有重要意義。

1 NLRP3炎癥小體的組成

NLRP3炎癥小體是一種存在于細胞質(zhì)中的蛋白復(fù)合物,由NLRP3、凋亡相關(guān)斑點樣蛋白(ASC)以及半胱天冬肽酶-1(caspase-1)前體蛋白結(jié)合而成。NLRP3由C端亮氨酸重復(fù)序列(LRR)和核苷酸寡聚化結(jié)構(gòu)域(NACTH)以及N端效應(yīng)結(jié)構(gòu)域(PYD)組成[28],其中PYD連接ASC的PYD結(jié)構(gòu)域后,ASC的C端CARD(caspase recruitment domain)結(jié)構(gòu)域招募caspase-1前體,進而激活caspase-1,實現(xiàn)炎癥小體NLRP3的裝配[29]。靜息狀態(tài)時,NLRP3處于無活性狀態(tài),只有被激活后才能轉(zhuǎn)化為炎癥小體發(fā)揮作用,具體的激活物分為病原相關(guān)分子模式(PAMPs)和損傷相關(guān)分子模式(DAMPs),其中PAMPS主要包括金黃色葡萄球菌、B組鏈球菌、李斯特氏菌、病毒[30-33]等;DAMPS主要包括尿酸或膽固醇結(jié)晶、b淀粉狀蛋白、三磷酸腺苷(ATP)等[34-36]。當PAMPs或DAMPs被Toll樣受體(第一信號)識別后,激活核轉(zhuǎn)錄因子NF-kB,啟動NLRP3、pro-IL-1b和pro-IL-18等的轉(zhuǎn)錄過程,進而上調(diào)炎癥小體的表達水平。第二信號(ATP,某些細菌毒素或顆粒物質(zhì))介導(dǎo)NLRP3的寡聚化,將NLRP3、ASC和caspase-1前體蛋白裝配成復(fù)合物,引起caspase-1的活化以及IL-1b和IL-18的分泌[37-38]。

2 NLRP3炎癥小體的激活途徑

目前,NLRP3炎癥小體主要存在4種激活路徑。①細胞外的ATP(三磷酸腺苷)充當NLRP3受體激動劑,通過結(jié)合表面受體P2X7,使離子通道打開。同時,細菌毒素使細胞膜形成空隙,從而誘導(dǎo)K﹢外流,從而導(dǎo)致NLRP3炎癥小體的組裝和激活?？梢哉J為,K﹢外流是NLRP3炎癥小體的主要催化劑,而細胞外ATP和成孔毒素是炎癥因子分泌的主要誘因[39]。②在PAMPs和DAMPs影響下,損傷的線粒體激發(fā)活性氧(ROS)、線粒體DNA和心磷脂的產(chǎn)生,作為激活NLRP3炎癥小體的關(guān)鍵信號導(dǎo)致NLRP3裝配和激活[40-41]。③結(jié)晶或顆粒結(jié)構(gòu)(如二氧化硅、石棉、淀粉樣蛋白b和明礬等)刺激吞噬細胞內(nèi)環(huán)境的穩(wěn)定,引起溶酶體破裂,組織蛋白酶B釋放到細胞質(zhì)中,直接激活炎性小體[42]。④Ca2﹢通量和Ca2﹢依賴性信號觸發(fā)NLRP3炎癥小體組裝[43-44]。

最近的研究表明,NIMA相關(guān)激酶(NEK)家族的成員NEK7直接與NLRP3的LRR結(jié)構(gòu)域結(jié)合,在K﹢外排和ROS產(chǎn)生作用下,完成炎癥小體的組裝與激活[45]。Wang W等[46]發(fā)現(xiàn)EV71(腸道病毒71)3D蛋白作為RNA依賴性RNA聚合酶,通過直接結(jié)合NLRP3刺激NLRP3炎性細胞的活化。除此之外,自噬功能障礙、線粒體鈣離子超載引起的功能失調(diào)、TXNIP的活化、PYD結(jié)構(gòu)域的磷酸化、高爾基體介導(dǎo)的蛋白激酶D信號傳導(dǎo)、氯離子等因素也可以激活NLRP3炎癥小體[47-51]。NLRP3炎癥小體組裝與激活途徑如圖1所示。

然而NLRP3炎癥小體的不適當激活會破壞先天性免疫應(yīng)答在人體的防御作用,研究表明,胰腺癌、動脈硬化、糖尿病、慢性肝病、帕金森等自身免疫相關(guān)性疾病都與其相關(guān)[52-56]。

3 NLRP3的負調(diào)控途徑

近幾年發(fā)現(xiàn),NLRP3的激活可以被自噬等多種調(diào)控途徑所抑制。自噬作為一種自我降解過程,在某些條件下,可以選擇性去除特定的蛋白質(zhì)和受損的細胞器(如線粒體)[57],從而維持細胞代謝平衡,實現(xiàn)內(nèi)環(huán)境穩(wěn)態(tài)。自噬由4個連續(xù)步驟組成,自噬體的形成,自噬膜的伸長和閉合,自噬體和溶酶體之間的融合以及降解,這種復(fù)雜過程在哺乳動物中至少由18個自噬相關(guān)基因(Atg基因)介導(dǎo)[58]。最近的研究已經(jīng)確定了自噬相關(guān)基因在NLRP3炎癥細胞活化中的關(guān)鍵作用,如Saitoh T等[59]發(fā)現(xiàn)Atg16L1可以通過促進自噬體P62的降解來抑制IL-1b的分泌;van der Burgh R等[60]發(fā)現(xiàn)Atg7缺陷導(dǎo)致線粒體受損,NLRP3炎癥體活化,與自噬可以通過清除損傷的線粒體直接抑制NLRP3炎癥體激活的結(jié)論一致[61]。有研究[61-62]發(fā)現(xiàn)3MA(PI3K抑制劑)抑制自噬增加了mtROS的產(chǎn)生,導(dǎo)致在沒有炎性體刺激的情況下,NLRP3依賴性IL-1b分泌;在干預(yù)結(jié)核分枝桿菌感染的細胞時,3MA阻斷自噬體形成導(dǎo)致IL-1b分泌增加。Spalinger MR等[63]發(fā)現(xiàn)PTPN22(蛋白酪氨酸磷酸酶,非受體型22)的喪失通過自噬途徑,增強的NLRP3磷酸化水平抑制NLRP3炎癥體的激活。除此之外,作為Fork框架轉(zhuǎn)錄因子家族的成員,Foxo3a在營養(yǎng)缺乏條件下參與自噬形成。研究證實,Foxo3a在細胞核中被抑制使自噬功能受損,NLRP3炎癥細胞在KCs中被激活;Foxo3a的過表達恢復(fù)自噬通量,并通過促進Bim的轉(zhuǎn)錄來減弱NLRP3炎癥細胞的活化[64]。

注:紅色箭頭與字體為第一信號,黑色為第二信號,①②③④為主要激活途徑

除自噬外,泛素相關(guān)蛋白可以負調(diào)控NLRP3的激活。E3泛素連接酶TRIM31(三分體基序蛋白31)被認為是NLRP3炎癥反應(yīng)抑制因子,它可以直接結(jié)合NLRP3,促進NLRP3的K48連鎖聚泛素化和蛋白酶體降解,同時,TRIM31缺乏可以減輕DSS誘導(dǎo)的結(jié)腸炎[65]。另外,TRIM30通過抑制細胞內(nèi)ROS的產(chǎn)生,從而抑制NLRP3炎癥小體激活[66]。

此外,NO與CO以及Nrf2(核轉(zhuǎn)錄因子E2相關(guān)因子2)通過抑制線粒體中ROS的產(chǎn)生,線粒體膜電位,和線粒體DNA在巨噬細胞中的胞內(nèi)易位,維持線粒體穩(wěn)定來抑制NLRP3的表達,ASC的裝配和IL-1b分泌達到負調(diào)控的作用[67-69]。研究發(fā)現(xiàn),NLRP3炎癥體激活物觸發(fā)Src同源2域包含蛋白酪氨酸磷酸酶-2(SHP2)易位到線粒體,與腺嘌呤核苷酸t(yī)ranslocase 1(ANT1)相互作用和去磷酸化抑制線粒體膜電位以及線粒體DNA和活性氧的釋放,從而防止NLRP3炎癥小體的過度激活[70]。

4 NLRP3炎癥小體與IBD的關(guān)系

近幾年的研究發(fā)現(xiàn),NLRP3在IBD的發(fā)病與炎癥中具有促進炎癥與維持腸道穩(wěn)態(tài)的雙重作用[3]。

4.1 NLRP3在IBD病理學中的促炎作用

研究顯示,在CD患者和患有急性或慢性結(jié)腸炎小鼠的結(jié)腸組織中存在NLRP3的高表達[33,71-73]。與NLRP3相關(guān)NLRs家族中的NOD1和NOD2存在于細胞質(zhì)中,它們分別識別細菌肽聚糖、胞壁酰二肽,Paneth細胞、上皮細胞和抗原呈遞細胞中的轉(zhuǎn)錄因子NF-kB和AP-1被活化,導(dǎo)致促炎因子IL-b的釋放[74]。DE la Fuente M等[75]發(fā)現(xiàn)從IBD患者分離的大腸桿菌菌株可以通過NLRP3炎癥小體誘導(dǎo)小鼠巨噬細胞的IL-1b分泌。Liu W等[76]使用NLRP3依賴的pro-caspase-1、IL-1b和IL-18的抑制劑Fc11a-2來治療模型小鼠,急性結(jié)腸炎的癥狀得到減輕。Bauer C等[77]發(fā)現(xiàn),與正常小鼠對比,NLRP3-/-小鼠在DSS造模的情況下,結(jié)腸炎的嚴重程度明顯下降。以上這些研究均提示,NLRP3對結(jié)腸炎的發(fā)生發(fā)展具有促進作用,且可以作為開發(fā)IBD患者的新型治療劑的潛在靶標。

4.2 NLRP3維持腸道內(nèi)穩(wěn)態(tài)作用

NLRP3在調(diào)節(jié)腸內(nèi)穩(wěn)態(tài)中起著關(guān)鍵作用,維持腸道上皮屏障完整性并降低實驗性結(jié)腸炎期間的死亡率,并影響腸道內(nèi)生物群的組成[48,78-80]。研究發(fā)現(xiàn)攜帶NLRP3 R258W突變的CAPS模型小鼠通過調(diào)節(jié)性T細胞(Treg細胞)誘導(dǎo)IL-1b的分泌增加,促進局部抗微生物肽促進微生物群的重建,從而維持腸道穩(wěn)態(tài)[81]。有學者[78-79,82]發(fā)現(xiàn)和NLRP3-/-、ASC-/-和caspase-1-/-小鼠對葡聚糖硫酸鈉(DSS)和2,4,6-三硝基苯磺酸(TNBS)誘導(dǎo)的結(jié)腸炎更易感染,小鼠腸道上皮細胞增殖缺陷,上皮屏障功能減弱,細菌的透過率升高,從而使易感性增加。除此之外,NLLRP3-/-小鼠在NO合成酶(iNOS)活性增加的誘導(dǎo)情況下,NO水平升高,IL-10和TGF-b表達降低,從而表明NLRP3可以在腸炎中下調(diào)炎癥反應(yīng),在腸內(nèi)穩(wěn)態(tài)中起關(guān)鍵作用[79]。在惡唑酮(OXA)誘導(dǎo)的結(jié)腸炎WT小鼠模型中,外源性IL-1b、IL-18可通過調(diào)節(jié)Th2細胞因子的表達,降低結(jié)腸炎的嚴重性。而NLRP3-/-小鼠結(jié)腸炎的嚴重性與Th2細胞因子的表達呈正相關(guān)。這提示NLRP3衍生的IL-1b、IL-18可以通過不同的機制抑制Th2細胞因子產(chǎn)生,減輕腸道損傷[83]。研究還發(fā)現(xiàn)NLRP3炎癥小體可以調(diào)節(jié)上皮細胞的衍生物IL-18的分泌,來維持腸道穩(wěn)態(tài)[84]。以上研究均提示NLRP3炎癥小體對實驗性結(jié)腸炎有保護作用。

隨著研究的深入,雖然研究者對NLRP3炎癥小體有了較多的認識,但其在IBD中的作用機制尚不明確,對于NLRP3炎癥小體表達水平對結(jié)腸炎的影響仍然存在爭議。特別是在NLRP3-/-小鼠結(jié)腸炎模型中的結(jié)果差異,可能與誘導(dǎo)結(jié)腸炎的方案、實驗條件、遺傳背景和所使用的動物的性別以及不同小鼠之間不同微生物群落等差異相關(guān)[85]。所以如要確定NLRP3炎癥小體在穩(wěn)定狀態(tài)條件下和在IBD中發(fā)揮的作用需要做進一步的研究。

5 針灸對炎癥性腸病NLRP3炎癥小體的作用與展望

目前,針灸對炎癥性腸病NLRP3炎癥小體是否有調(diào)節(jié)作用尚未見有報道,但業(yè)已證實針灸對NLRP3信號通路的激活因素、下游炎癥因子以及負調(diào)控的相關(guān)因素,有一定的調(diào)節(jié)作用,提示調(diào)節(jié)NLRP3炎癥小體及相關(guān)信號通路可能是針灸治療IBD的重要效應(yīng)機制。

NF-kB與NLRP3炎癥小體的組裝與激活相關(guān),研究發(fā)現(xiàn)針灸對IBD核轉(zhuǎn)錄因子NF-kB及下游炎癥因子有調(diào)節(jié)作用。

在克羅恩病中,研究發(fā)現(xiàn)電針刺激足三里、曲池穴能夠減輕TNBS誘導(dǎo)的結(jié)腸病變,與其下調(diào)IL-1b的表達,以及抑制p-IkBa和NF-kB p65等蛋白表達水平相關(guān)[86];艾灸天樞、氣海穴,能下調(diào)克羅恩病大鼠結(jié)腸NF-kB p65、TNF-a、IL-1b的表達,提示艾灸可能通過抑制克羅恩病大鼠結(jié)腸NF-kB p65的表達,進而減少其下游炎癥因子TNF-a、IL-1b的表達,從而減輕腸道炎癥,改善結(jié)腸組織形態(tài)結(jié)構(gòu),發(fā)揮治療作用[87]。在潰瘍性結(jié)腸炎中,研究發(fā)現(xiàn),電針上巨虛能調(diào)節(jié)潰瘍性結(jié)腸炎IL-1b的異常表達,改善結(jié)腸黏膜損傷[88];大鼠潰瘍性結(jié)腸炎的發(fā)生涉及多種基因的表達異常,隔藥灸可通過調(diào)節(jié)IL-1b等諸多基因表達起到治療作用[89]。艾灸可通過抑制結(jié)腸組織NF-kB p65轉(zhuǎn)錄而下調(diào)TLR-9表達,從而修復(fù)UC大鼠受損黏膜上皮,抑制血清中IL-8含量,提高血清中IL-10含量,且灸量越大效果越明顯[90]。還有研究發(fā)現(xiàn)調(diào)控TLR/MyD88/NF-kB信號轉(zhuǎn)導(dǎo)通路中MyD88、TRAF6的表達是隔藥灸調(diào)節(jié)潰瘍性結(jié)腸炎結(jié)腸黏膜局部過激免疫炎癥反應(yīng)的作用途徑之一[91]。

除此之外,針灸對NLRP3負調(diào)控途徑的相關(guān)因素也有一定的作用。有研究發(fā)現(xiàn),在金黃色葡萄球菌引起的巨噬細胞感染中,溫和灸可以下調(diào)IL-1b、iNOS以及caspase-1的表達,減輕炎癥;除此之外還證實了,溫和灸是通過下調(diào)自噬mTOR通路中Akt的磷酸化水平來起到作用的[92]。

綜上所述,針灸對NLRP3炎癥小體下游炎癥因子、調(diào)控途徑均有一定的調(diào)節(jié)作用。NLRP3炎癥小體作為系列炎癥反應(yīng)的核心,可能是針灸防治炎癥性腸病新的重要靶點。探討艾灸調(diào)節(jié)炎癥小體及其相關(guān)信號通路治療炎癥性腸病的效應(yīng)機制,對于炎癥性腸病的防治至關(guān)重要。以NLRP3炎癥小體為介入點,觀察針灸對炎癥性腸病NLRP3炎癥小體及上下游通路信號分子的調(diào)節(jié)作用,闡釋針灸對NLRP3炎癥小體調(diào)控的分子機制,有助于進一步闡明針灸防治炎癥性腸病的作用機制,為臨床炎癥性腸病的針灸治療提供科學證據(jù)。

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Research Advances in the Relationship Between NLRP3 Inflammasome and Inflammatory Bowel Disease and the Regulating Effect of Acupuncture and Moxibustion

1,1,2,1,2,1,2,2,1,2.

1.201203,; 2.200030,

Inflammatory bowel disease (IBD) is a group of chronic nonspecific inflammatory intestinal diseases including Crohn's disease (CD) and ulcerative colitis (UC). The inflammasome is macromolecular multiprotein and plays an important role in inflammatory occurrence and development and bodily innate immunity. NLRP3 inflammasome produces a bidirectional regulating effect in the occurrence and development of IBD and the mechanism of maintaining intestinal homeostasis. This article reviews research advances in NLRP3 inflammasome in IBD, and the regulating effect of acupuncture and moxibustion on it and the possible mechanism to provide a new idea for clinical and mechanism studies of acupuncture-moxibustion treatment for inflammatory bowel disease.

Inflammatory bowel disease; Acupuncture-moxibustion therapy; Inflammasome; NLRP3

1005-0957(2018)08-0961-08

R2-03

A

10.13460/j.issn.1005-0957.2018.08.0961

2018-03-20

國家自然科學基金項目(81674073,81273843);國家重點基礎(chǔ)研究發(fā)展計劃項目(2015CB554501);上海市衛(wèi)生和計劃生育委員會項目(2017BR047)

吳麗潔(1990—),女,2017級博士生,Email:wljzwb@163.com

馬曉芃(1973—),女,研究員,博士生導(dǎo)師,Email:pengpengma@163.com