丁佳，吳健

復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院醫(yī)學(xué)分子病毒學(xué)教育部/衛(wèi)生部重點(diǎn)實(shí)驗(yàn)室，上海 200032

隨著肥胖、2型糖尿病和代謝綜合征的發(fā)病率逐年升高，非酒精性脂肪性肝病(non-alcoholic fatty liver disease，NAFLD)已成為20世紀(jì)最常見的慢性肝臟疾病之一。NAFLD包括單純性肝脂肪變、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis，NASH)伴或不伴肝纖維化，后者可進(jìn)展為終末期肝病(肝硬化)，并在此基礎(chǔ)上發(fā)生肝細(xì)胞肝癌(簡(jiǎn)稱肝癌)。NASH已成為近年來西方國(guó)家肝癌發(fā)病率升高的主要因素之一。除遺傳基因、飲食環(huán)境和生活方式等因素與NAFLD的發(fā)病相關(guān)外，近期研究表明腸道菌群組分改變?cè)谡T發(fā)能量代謝紊亂和胰島素抵抗，促進(jìn)NAFLD發(fā)展至NASH中也起重要作用。本文旨在總結(jié)近年來在探索腸-肝軸和腸道菌群改變與肝臟炎癥發(fā)生、發(fā)展關(guān)系方面的研究進(jìn)展，為擬定NASH治療的新策略提供科學(xué)依據(jù)。

1 腸-肝軸相互作用

腸道菌群對(duì)肝臟疾病的影響至少可追溯到80年前。Hoefert首先報(bào)道小腸細(xì)菌過度生長(zhǎng)普遍出現(xiàn)于肝硬化患者，并與疾病的嚴(yán)重程度直接相關(guān)[1]。由于解剖結(jié)構(gòu)和位置的特殊性，肝臟作為一級(jí)淋巴器官直接處理腸道血流中大量的細(xì)菌內(nèi)毒素和代謝產(chǎn)物，通過膽鹽等底物和腸-肝循環(huán)調(diào)節(jié)腸道功能、激素和免疫反應(yīng)。同時(shí)，由于肝臟與腸道間的雙向作用，腸道的激素、炎性介質(zhì)和消化吸收產(chǎn)物也能調(diào)節(jié)肝功能。肝硬化時(shí)小腸動(dòng)力障礙和口-盲腸傳輸時(shí)間延長(zhǎng)是小腸細(xì)菌過度生長(zhǎng)的生理基礎(chǔ)，同時(shí)腸道黏膜通透性增加為腸道內(nèi)細(xì)菌過度生長(zhǎng)、侵入腹腔創(chuàng)造了條件，從而更易發(fā)生自發(fā)性細(xì)菌性腹膜炎和肝性腦病[2,3]。這是腸-肝軸影響肝臟疾病發(fā)生、發(fā)展的典型案例。

2 腸道菌群改變影響NAFLD的發(fā)生

在人類的消化道棲息著億萬(wàn)種不同種類的微生物，其中大部分細(xì)菌種系定植在結(jié)腸，細(xì)菌總數(shù)為1013～1014個(gè)，菌體量> 1 kg。細(xì)菌的數(shù)量和類別保持動(dòng)態(tài)恒定是維持腸道內(nèi)環(huán)境穩(wěn)定的基礎(chǔ)[4]。人類宏基因組包括嵌入人體的基因組和伴生的微生物基因組。作為一個(gè)整體，微生物基因組數(shù)量是人類基因組數(shù)量的100余倍[5]。消化道在出生時(shí)是無(wú)菌的，出生后細(xì)菌開始定植并發(fā)展成穩(wěn)定的菌群。嬰兒腸道中的雙歧桿菌是分解母乳中寡糖的關(guān)鍵菌群，能否建立穩(wěn)定的菌群是嬰兒正常消化吸收功能的重要前提。嬰幼兒腸道菌群不穩(wěn)定是發(fā)生腹瀉和消化吸收障礙的原因之一[6]。成年后消化道內(nèi)菌群的數(shù)量和類別保持相對(duì)穩(wěn)定，但個(gè)體間差異巨大。消化道不同解剖部位的細(xì)菌種類和數(shù)量也不同，升結(jié)腸的細(xì)菌含量為1011個(gè)細(xì)胞/g，遠(yuǎn)端回腸為107～108個(gè)細(xì)胞/g，近端空回腸為102～103個(gè)細(xì)胞/g[7]。盡管腸道菌群的種系估計(jì)超過5 000種，但只有少部分菌種在數(shù)量上占主導(dǎo)地位[8,9]，如厚壁菌門約占60%、擬桿菌門約占15%、放線菌門約占15%、疣微菌門約占2%、變性菌門約占1%、甲烷桿菌目約占1%[10]。其中，擬桿菌門合成氫氣，厚壁菌門合成各種可作為人體能量來源的短鏈脂肪酸(short chain fatty acid，SCFA)，包括丁酸等。此外，同一門類中的微生物功能可具有高度多樣化[11]。

研究表明，超重和肥胖兒童糞便中雙歧桿菌比例下降，金黃色葡萄球菌比例上升[12]。與健康人群相比，肥胖人群體內(nèi)厚壁菌門比例上升，而擬桿菌門比例下降[13]。某些腸道菌群能更高效地從食物中攝取能量，促進(jìn)肥胖和NAFLD的發(fā)生。Miele等對(duì)35例組織學(xué)證實(shí)的NAFLD患者研究后發(fā)現(xiàn)，患者腸道黏膜通透性增加，黏膜上皮細(xì)胞間緊密連接缺失，帶狀閉合蛋白1(zona occludens 1，ZO-1)表達(dá)下調(diào)。其中60%患者出現(xiàn)小腸細(xì)菌過度生長(zhǎng)，且與肝脂肪變的嚴(yán)重程度顯著相關(guān)[14]。動(dòng)物實(shí)驗(yàn)表明，高脂飼料喂養(yǎng)的小鼠糞便中厚壁菌門比例升高，腸道內(nèi)SCFA合成增加[15]。SCFA作為機(jī)體重要的能量來源，能增強(qiáng)肝臟自身脂質(zhì)合成和三酰甘油聚集；同時(shí)也是腸上皮細(xì)胞、腸內(nèi)分泌細(xì)胞和脂肪細(xì)胞的G蛋白偶聯(lián)受體(G protein-coupled receptor，GPCR)的配體[16]。通過與GPCR結(jié)合，SCFA可增強(qiáng)胃腸激素如胰高血糖素樣肽1(glucagon-like peptide 1，GLP-1)和酪酪肽(peptide YY，PYY)的分泌，直接或間接影響胰島素和胰高血糖素的產(chǎn)生，調(diào)節(jié)食欲和食物的攝入[17]。

此外，動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)部分高脂飼料喂養(yǎng)的小鼠(稱為“應(yīng)答”小鼠)除體重增加外，空腹血糖和胰島素水平都升高，伴有單核細(xì)胞趨化因子1(monocyte chemoattractant protein 1，MCP-1)和腫瘤壞死因子α(tumor necrosis factor α，TNF-α)等促炎因子分泌增加。將“應(yīng)答”小鼠和“非應(yīng)答”小鼠的腸道菌群分別移植至無(wú)菌小鼠后，接受“應(yīng)答”小鼠腸道菌群接種的受體小鼠出現(xiàn)了脂肪肝，并伴有轉(zhuǎn)錄因子固醇調(diào)節(jié)元件結(jié)合蛋白1c(sterol regulatory element binding protein 1c，SREBP1c)、糖類應(yīng)答元件結(jié)合蛋白(carbohydrate response element binding protein，ChREBP)和乙酰輔酶A 羧化酶等脂質(zhì)合成限速酶基因表達(dá)上調(diào)；而“非應(yīng)答”受體小鼠則無(wú)顯著變化。與“非應(yīng)答”受體小鼠相比，“應(yīng)答”受體小鼠糞便內(nèi)厚壁菌門比例顯著升高，表明腸道菌群的改變促進(jìn)NAFLD發(fā)生[18]。但此結(jié)果能否在人體得到證實(shí)，仍需多樣本臨床對(duì)照研究驗(yàn)證。由于厚壁菌門被認(rèn)為是“肥胖菌群”，且能在同一種系間接種傳播，故有學(xué)者將由此類細(xì)菌引起的能量代謝異常、肥胖和NALFD定義為“感染性疾病”[19]。這一論點(diǎn)能否得到公認(rèn)，有待商榷。

3 腸道菌群調(diào)控肝臟炎癥和肝纖維化

腸道細(xì)菌的產(chǎn)物，如脂多糖(lipopolysaccharide，LPS)、脂多肽、DNA和RNA，具有潛在的肝毒性，能促進(jìn)炎癥發(fā)生[20]。這些細(xì)菌產(chǎn)物通過機(jī)體天然免疫系統(tǒng)的病原體相關(guān)分子模式(pathogen-associated molecular pattern，PAMP)和損傷相關(guān)分子模式(damage-associated molecular pattern，DAMP)激活肝細(xì)胞、Kupffer細(xì)胞和肝星狀細(xì)胞(hepatic stellate cell，HSC)表面和細(xì)胞內(nèi)的Toll樣受體(Toll-like receptor，TLR)，啟動(dòng)相應(yīng)的炎癥反應(yīng)[21]。不同的TLR具有相應(yīng)的配體和特異的PAMP和DAMP[22]。TLR是進(jìn)化高度保守的Ⅰ型跨膜糖蛋白，包含2個(gè)結(jié)構(gòu)域：富含亮氨酸的重復(fù)序列和細(xì)胞內(nèi)信號(hào)結(jié)構(gòu)域——Toll/白細(xì)胞介素1受體(Toll/interleukin 1 receptor，TIR)結(jié)構(gòu)域[23]。TLR下游的信號(hào)通路包括髓樣分化因子88(myeloid differentiation factor 88，MyD88)依賴或MyD88非依賴2條通路。MyD88依賴的下游信號(hào)通路激活核因子κB(nuclear factor κB，NF-κB)，促進(jìn)TNF-α、白細(xì)胞介素6(interleukin 6，IL-6)、IL-8和IL-12等促炎因子和γ干擾素(interferon γ，IFN-γ)、MCP-1等免疫相關(guān)基因和趨化因子的轉(zhuǎn)錄；MyD88非依賴的信號(hào)通路主要促進(jìn)下游IFN-β的表達(dá)[24]。其中，TLR4能通過與配體革蘭陰性桿菌胞壁成分LPS結(jié)合，同時(shí)以MyD88依賴和MyD88非依賴2種途徑激活下游信號(hào)通路[25]。

在小鼠NAFLD模型中，將普通飼料換成高脂飼料后，小鼠腸道菌群中厚壁菌門比例升高，擬桿菌門比例下降。小鼠體重增加，空腹血糖和胰島素水平升高，肝內(nèi)三酰甘油和炎性分子聚集，門靜脈中LPS水平顯著升高[26]。Henao-Mejia等研究表明，IL-1細(xì)胞因子超家族成員IL-18能通過調(diào)節(jié)腸道菌群，在NAFLD等代謝性疾病進(jìn)展中起重要作用。IL-1β和IL-18被炎性小體復(fù)合物激活后，才能形成有生物學(xué)活性的分子。炎性小體復(fù)合物由半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase 1, caspase-1)和核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體(nucleotide-binding oligomerization domain-like receptor，NLR)家族蛋白組成。NLRP3和NLRP6天然免疫缺陷小鼠失去產(chǎn)生IL-18的能力，腸道菌群譜中普雷沃菌科和紫單胞菌科比例增加，導(dǎo)致細(xì)菌產(chǎn)物由腸道轉(zhuǎn)位至血循環(huán)和肝臟，激活TLR4和TLR9。該小鼠在喂食膽堿缺失飼料后極易發(fā)生NAFLD，喂食高脂飼料后極易發(fā)生代謝綜合征[27]。

NAFLD患者小腸細(xì)菌過度生長(zhǎng)、小腸黏膜通透性增加及緊密連接丟失引起的細(xì)菌轉(zhuǎn)位導(dǎo)致血漿中LPS濃度升高，這些改變與NAFLD向NASH進(jìn)展相關(guān)[14,28,29]。與輕度肝脂肪變患者相比，中、重度肝脂肪變患者的腸道黏膜通透性增加，小腸細(xì)菌過度生長(zhǎng)的程度更甚[30]。NASH患者小腸細(xì)菌過度生長(zhǎng)的嚴(yán)重程度與TLR4表達(dá)增強(qiáng)和IL-8生成密切相關(guān)[29]。肝組織炎癥時(shí)，Kupffer細(xì)胞和HSC中TLR4表達(dá)水平升高。Kupffer細(xì)胞首先對(duì)LPS作出反應(yīng)，產(chǎn)生炎性細(xì)胞因子、趨化因子和活性氧(reactive oxygen species，ROS)[31]，并通過分泌轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor β，TGF-β)等促纖維化因子激活HSC[32]。與健康人群相比，NASH患者小腸細(xì)菌過度生長(zhǎng)引起血漿中LPS增多，經(jīng)LPS-TLR4信號(hào)途徑誘導(dǎo)TNF-α水平顯著升高[33,34]。除通過Kupffer細(xì)胞間接激活HSC外，腸道來源的LPS可直接通過TLR4激活HSC。LPS通過下調(diào)TGF-β負(fù)向調(diào)節(jié)受體Bambi的功能，強(qiáng)化TGF-β對(duì)HSC的激活，活化的HSC分泌CC趨化因子配體2(CC chemokine ligand 2，CCL2)和CCL4等趨化因子，招募Kupffer細(xì)胞，后者再分泌TGF-β，繼而促進(jìn)肝纖維化進(jìn)展[32]。如此，LPS所致肝內(nèi)非實(shí)質(zhì)細(xì)胞間相互作用使肝臟炎癥和纖維化反應(yīng)得以持續(xù)。

其他腸道細(xì)菌產(chǎn)物如細(xì)菌DNA，可通過DAMP模式影響NASH的進(jìn)展和慢性化。細(xì)菌DNA富含CpG序列，與細(xì)胞內(nèi)TLR9結(jié)合后，通過MyD88招募和激活下游NF-κB等信號(hào)分子，促進(jìn)炎癥分子表達(dá)[35]。此外，TLR9還通過干擾素調(diào)控因子7(interferon regulatory factor 7，IRF-7)促進(jìn)IFN-α表達(dá)[36]。肝細(xì)胞、肝血竇內(nèi)皮細(xì)胞、Kupffer細(xì)胞和HSC都可功能性表達(dá)TLR9[37,38]。小鼠NASH模型中，細(xì)菌DNA通過與Kupffer細(xì)胞內(nèi)TLR9結(jié)合，以MyD88依賴途徑促進(jìn)IL-1β表達(dá)。IL-1β通過活化HSC，上調(diào)Ⅰ型前膠原和金屬 蛋白酶組織抑制劑1(tissue inhibitor of metalloproteinase 1，TIMP-1)等促纖維化基因表達(dá)及下調(diào)Bambi表達(dá)，促進(jìn)肝纖維化。TLR-9缺陷和IL-1受體缺陷小鼠與野生型小鼠相比，脂肪性肝炎和肝纖維化程度顯著下降[39]。

4 腸-肝軸可作為防治NASH和肝纖維化的新策略

肥胖和高脂飲食可改變腸道菌群，因此可通過抗生素、益生菌和益生元等藥物調(diào)節(jié)腸道菌群，改善腸道黏膜屏障，抑制小腸細(xì)菌過度生長(zhǎng)，降低外周血和門靜脈內(nèi)毒素水平，以達(dá)到控制NASH向肝纖維化進(jìn)展的目標(biāo)。

動(dòng)物實(shí)驗(yàn)表明，雙歧桿菌可改善高脂飼料小鼠的葡萄糖穩(wěn)態(tài)，降低小鼠體重和體脂含量，恢復(fù)葡萄糖介導(dǎo)的胰島素分泌，并降低促炎性細(xì)胞因子(如TNF-α、IL-6等)和LPS水平[40]。給予ob/ob小鼠口服益生菌復(fù)合制劑VSL#3(唾液鏈球菌、嗜熱鏈球菌、雙歧桿菌和嗜酸乳酸桿菌等8種活性益生菌混合物)，可降低肝內(nèi)脂質(zhì)含量和肝臟炎癥，改善肝臟的胰島素抵抗[41]，并能通過下調(diào)NF-κB活性降低促炎性細(xì)胞因子分泌，以及降低TNF-α、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase，iNOS)和環(huán)氧酶2等脂質(zhì)過氧化標(biāo)記的表達(dá)[42]。此外，VSL#3還可通過減少平滑肌α肌動(dòng)蛋白聚集、降低前膠原α1表達(dá)、刺激Bambi受體表達(dá)、控制HSC活化等機(jī)制，延緩膽堿缺乏飼料誘導(dǎo)的NASH纖維化進(jìn)程[43]。盡管益生菌在動(dòng)物模型中能緩解肝臟炎癥和降低肝內(nèi)脂肪聚積，但用其治療NAFLD的臨床試驗(yàn)結(jié)果卻不甚理想。因NAFLD患者在停藥4個(gè)月后肝內(nèi)脂質(zhì)含量顯著增加，故VSL#3治療NAFLD的臨床試驗(yàn)被提前終止[44]。

口服腸道不吸收抗生素可調(diào)節(jié)腸道菌群，但由于無(wú)法特異性針對(duì)“有害”細(xì)菌，故備受爭(zhēng)議。研究表明，給予高脂飼料誘導(dǎo)的肥胖小鼠口服萬(wàn)古霉素，可使厚壁菌門與擬桿菌門細(xì)菌比例顯著下降，變形菌門細(xì)菌數(shù)量降低。在萬(wàn)古霉素干預(yù)期間，盡管進(jìn)食同等熱量的食物，小鼠體重較對(duì)照組降低，空腹血糖、血漿TNF-α及三酰甘油水平顯著下降[45]。在果糖誘導(dǎo)的小鼠NASH模型中，口服不吸收抗生素可顯著降低血漿和門靜脈LPS水平及肝臟TNF-α表達(dá)，減緩肝細(xì)胞脂肪變和肝臟炎癥損傷[43]。但迄今為止，尚未有研究肯定益生菌或抗生素能顯著減緩NASH患者向肝纖維化及終末期肝病進(jìn)展。

腸道菌群與NAFLD的關(guān)系強(qiáng)調(diào)了腸-肝軸相互作用對(duì)機(jī)體能量代謝的影響。鑒于一些腸道細(xì)菌的組分和產(chǎn)物與NAFLD進(jìn)展至NASH相關(guān)，人們?cè)噲D通過定性和定量改變腸道菌群以延緩肝臟疾病的進(jìn)程。盡管動(dòng)物模型和部分臨床研究顯示，益生菌對(duì)NASH患者的肝臟損傷有潛在的治療作用，但迄今尚無(wú)大規(guī)模的臨床隨機(jī)對(duì)照試驗(yàn)來驗(yàn)證這一結(jié)果。腸道菌群的類型和功能鑒定、小腸細(xì)菌過度生長(zhǎng)與NASH起始和進(jìn)展的因果關(guān)系，仍有待進(jìn)一步研究。同時(shí)，NASH由胰島素抵抗、肝脂質(zhì)毒性、氧化應(yīng)激反應(yīng)等多因素引起，且常為肥胖、糖尿病及代謝綜合征等疾病的肝臟表現(xiàn)或伴生疾病。NASH發(fā)病機(jī)制的個(gè)體化差異較大，一種機(jī)制往往不能解釋所有患者的發(fā)病原因，故其治療更應(yīng)趨于個(gè)體化[46]。

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