LPS/TLR4信号通路在肝胆管结石病中的作用及机制研究进展

唐世芳　综述　赵礼金　审校

（遵义医学院 1.临床学院　2.附属医院　肝胆外科，贵州 遵义 563000）

肝胆管结石病具有高发病率的特征，而目前尚无特效药。因此，导致较多肝胆管结石患者由于缺乏有效治疗导致胆汁淤积，发展成肝硬化。并且研究[1]发现，肝胆管结石病与肝内胆管癌的发生发展密切相关，是肝胆管良性疾病中引起患者死亡的最主要因素之一[2]。胆道感染是该病重要的发病机制之一，而近年有研究发现胆道感染能激活炎症信号通路，产生炎症瀑布效应从而引起慢性持续性炎症[3]。

近年来，越来越多的研究[4-6]明确了Toll样受体在免疫，特别是在感染免疫中发挥了重要的作用。Toll样受体家族（Toll-like receptors，TLRs）是最早发现的天然免疫模式识别受体（pattern recognition receptors，PRRs）[7]，通过识别外源配体的病原体相关分子模式（pathogen-associated molecular patterns，PAMPs）、内源性配体的损伤相关分子模式（damage-associated molecular patterns，DAMPs）以及异源物相关分子模式（xenobiotic-associated molecular patterns，XAMPs）来刺激先天免疫应答，同时还能通过获得性免疫来对机体进行保护[4,8-12]，但是这些应答所致的持续性炎症反应会对机体产生损伤，被认为是多种慢性疾病的触发因素[4]。越来越多的研究[4-6]明确了Toll样受体在免疫，特别是在感染免疫中发挥了重要的作用。Toll样受体4（TLR4）是人类发现的第一个Toll样受体相关蛋白，也是目前研究最广泛的Toll样受体相关蛋白之一，几乎分布于所有的细胞系。有研究[13-20]发现哺乳动物胆管上皮细胞、肝细胞、肾小管上皮细胞、肠上皮细胞等等均有表达TLR4。内毒素/脂多糖（lipopolysaccharide，LPS）是革兰氏阴性菌细胞壁的主要成分，是TLR4的天然配体。血液中的LPS主要通过两种方式激活TLR4信号通路：一是LPS被其结合蛋白LBP运送到细胞膜表面与该处TLR4的结构辅助蛋白CD14形成一个复合体，再同TLR4/MD-2相互作用，激活TLR4的下游信号通路；二是LPS直接与TLR4的附属蛋白MD-2结合并相互作用，进而激活TLR4下游信号通路[21]。TLR4信号转导通路由细胞内的TIR结构域启动[22]，目前研究发现的能够与TIR结构域结合的胞内衔接蛋白有髓样分化因子88（myeloid differentiation factor 88，MyD88）、TIR结构域接合子蛋白/MyD88接合子样蛋白（Toll-interleukin 1 receptor domain- containing adapter protein/MyD88 adaptor-like protein，TIRAP/Mal）、β干扰素TIR结构域衔接蛋白（TIR-domain-containing adaptor inducing interferon-β，TRIF）和TRIF相关接头分子（TRIF-related adaptor molecule，TRAM）[23]，TLR4信号传导主要通过Myd88依赖性通路和Myd88非依赖性通路即TRIF依赖性通路两种途径进行[24]。Myd88依赖性通路中Myd88激活信号转导因子包括白介素1受体联合激酶4（IL-1R-associated kinase 4，IRAK4）、肿瘤坏死因子受体相关因子6（TNF receptor-associated factor 6，TRAF6）和激活胞膜激酶（TGF-activated kinase 1，TAK1），激活下游的抑制IκB激酶（inhibitory κB kinase，IKK）和促分裂原活化蛋白激酶（mitogen-activated protein kinases，MAPK）通路，最后导致核转录因子NF-κB活化和相关促炎因子的产生。此外，干扰素调节因子5（interferon regulatory factor 5，IRF5）也被发现与Myd88通路有关[25]。TRIF依赖的Myd88非依赖性通路，能够激活干扰素调节因子3（interferon regulatory factor 3，IRF3）和TANK联合激酶1（TANK binding kinase1，TBK1）等信号转导分子，最终诱导干扰素β（interferon-β，IFN-β）的表达，引起相关的炎症反应[23]，研究[26]表明TRIF依赖通路也可以激活NF-κB和MAPK。

近年来研究[1]发现LPS/TLR4信号通路激活后引起持续炎性损伤可导致胆管上皮细胞（bile duct epithelia cells，BDECs）增殖，同时上皮细胞获得间质细胞的表型和功能，发生上皮-间质转化（epithelial-to-mesenchymal transition，EMT），参与肝胆管纤维化的进程。从而改变胆管树内胆汁流,导致形成结石胆汁的产生和分泌[27]。

1　肝胆管结石病发病机制概述

肝胆管结石也称作原发性肝内胆管结石，是指左右肝管汇合处以上的所有胆管内的胆结石。肝胆管结石的发病率有较大的地区差异，亚洲国家发病率远高于西方国家，尤其在东亚地区发病率非常高，在日本、韩国、中国发病率占肝胆系统疾病的1/4[1,28]。临床研究[27]表明，结石形成的最主要的病因是胆道感染，胆汁淤积是结石形成的必要条件。

2　LPS/TLR4和胆道感染

胆道感染是肝胆管结石病关键的发病机制，可导致病原相关分子或病原体产生LPS引发菌血症[29]，但引起胆道感染的相关机制尚未明确，相关研究表明，发现LPS/TLR4信号通路被激活后介导下游的信号传导，是胆道感染的相关机制之一。

2.1　LPS/TLR4与肠源性内毒素

肠道胆盐缺乏、小肠黏膜屏障损伤后，细菌会通过肠壁吸收进入门静脉移位于胆管，在胆汁中生长繁殖造成胆源性感染[30-32]，肝胆管结石患者几乎都存在胆道感染。临床研究[33]发现，肝胆管结石患者胆汁中检测出大量革兰阴性菌，其代谢产生的LPS的含量高低与胆管结石患者感染的程度正相关。目前对LPS-TLR4-NF-κB经典炎症通路的研究集中在人体其他正常组织和肿瘤组织的体外培养[34-36]。肠源性内毒素的大量产生会刺激胆道感染的反复发生，导致肝内胆管多发结石可引起肝损伤。大量产生的LPS可激活枯否细胞（kupffer cells，KCs）NF-κB并促使KCs释放高水平的TNFα、IL-6及IL-1等炎症因子，TNFα和IL-6过量表达，导致肝细胞凋亡和坏死，IL-1的升高又进一步抑制肝细胞再生[37]。因此，适当调控KCs NF-κB的活性有促进肝细胞再生的可能。

2.2　LPS/TLR4与KCs

KCs是位于肝窦隙内的巨噬细胞，是肝内固定的单核-巨噬细胞群，同时也是清除来自胆肠道内的细菌以及其产生的LPS的主要场所[38]。胆道感染时，LPS激活KCs合成并产生多种促炎因子[39]。CD14是存在于单核-巨噬细胞膜表面的LPS受体，可启动LPS介导的信号通路传导，研究[21]发现在生理状态下，少量LPS不会引起KCs细胞膜表面的CD14的表达或只有少量表达，但是异常的高浓度LPS能导致CD14的高表达，从而诱导炎症反应。CD14在LPS介导KCs分泌各种细胞因子的过程中扮演着十分重要的角色[40]。由于CD14是TLR4激活所必需的因子，胆道感染时，LPS与KCs细胞膜表面CD14的结合可能会通过激活LPS/TLR4信号通路从而介导了KCs合成和分泌。

2.3　LPS/TLR4与氧化应激

氧化应激是指机体内自由基产生过多，使机体的清除能力负荷超出正常水平，打破氧化/抗氧化平衡。研究表明氧化应激可导致脏器组织氧化损伤[41-42]。LPS通过与多种炎症细胞或效应细胞膜上的TLR4蛋白结合诱导产生某些炎症介质和细胞因子，激活细胞内MAPKs-NF-κB信号通路，致使产生炎性介质大爆发的瀑布效应，最终导致自由基产生[43-45]，引起氧化应激反应，加重肝胆管组织炎性损伤[46]。胆道感染时血液中氧、羟自由基增加会加速胆道内胆红素钙结石生成，沉淀颗粒增大，进而形成肝胆结石，再次加重胆道感染，形成恶性循环[27]。另一方面，胆道梗阻或者门静脉内毒素血症发生时肝脏内氧自由基增加，进一步损害KCs的清除能力[47]，从而加重胆道感染促进结石的形成。因此，在梗阻性黄疸的动物模型的胆道中应用内支架引流减压，可促使KCs清除能力的恢复[48]。

2.4　LPS/TLR4与葡萄糖醛酸酶mRNA的表达

LPS进入肝胆系统后可以刺激肝脏细胞、胆管上皮细胞及胆汁中白细胞分泌内源性β-葡萄糖醛酸酶（β-glucuronidas，β-GD）[49]。β-GD可以分解胆红素双葡糖醛酸酯，使结合胆红素分解成游离胆红素，游离胆红素又与钙结合生成胆红素钙结石[49]，参与肝胆结石的形成。LPS通过与肝脏细胞、胆管上皮细胞膜上的CD14结合后启动信号传导功能，通过LPS-TLR4-NF-κB信号通路启动细胞内控制β-GD的基因，由此转录更多的mRNA，增加β-GD蛋白质的合成。实验研究证明LPS可以使组织源性β-GD的合成和释放增加，可能有助于解释不伴有细菌感染的胆色素结石的发病原因[50]。

3　LPS/TLR4与胆汁淤积

TNF-α是由巨噬细胞、内皮细胞和库普弗细胞释放的细胞因子，是LPS/TLR4信号通路中引起全身效应的主要调节因子[51]。研究[52]发现，小鼠注射LPS后，离体灌注的肝脏胆汁流量发生下降，用抗TNF-α的抗体阻断后胆汁流量与胆盐分泌减少，提示TNF-α与LPS/TLR4信号通路诱导的胆汁淤积有关。LPS/TLR4信号通路参与胆囊炎症的发生和发展，导致胆囊功能受到影响，引起肝内胆管胆汁淤积，从而形成肝胆管胆石[53]。已有证据[27]证实，胆汁淤积是造成肝胆管结石的必要条件，因此推测，LPS/TLR4信号通路通过对胆管树的调节从而参与了肝胆管结石形成的发生。

4　LPS/TLR4与EMT

EMT是指具有极性、黏附性的上皮细胞表型转化成具有非极性、可自由移动且缺乏细胞间连接的间质细胞表型[54]。EMT参与胚胎形成、组织细胞修复和再生等生理过程，创伤后正常的纤维瘢痕修复过程持续存在时，就会发生非正常的病理过程导致多种组织的纤维化、硬化[55]。有学者[56-57]研究发现持续的炎性损伤可使胆管上皮细胞（bile duct epithelia cells，BDECs）增殖，上皮细胞获得间质细胞表型及功能，发生EMT，并参与肝胆管纤维化进程。同时，也有体外实验证实LPS能刺激BDECs发生EMT[58]。肝胆管结石病患者肝组织中的小胆管的BDECs发生增殖，通过EMT样现象或与肌纤维母细胞相互作用，促进疾病的发生发展[59]。TLR4参与活化并激活由LPS介导的肝内胆管上皮细胞发生上皮-间质转化[60]。体外研究[61]结果证明，对人肝内胆管上皮细胞（human intrahepatic biliary epithelial cells，HIBEpiC）进行沉默TLR4基因表达的转染实验，有效的抑制LPS诱导的肝内胆管上皮细胞发生上皮-间质转化，并且沉默TLR4表达后的HIBEpiC中检测发现上皮标志物表达明显升高，说明有效抑制了HIBEpiC发生EMT。TLR4可作为早期调控肝内胆管上皮细胞上皮-间质转化、抑制胆道纤维化进程的药物治疗靶点。因此，探索肝胆管结石病是否通过EMT导致肝胆管纤维化以及其全面的调节机制迫在眉睫。

5　结论与展望

LPS/TLR4信号通路在肝胆管结石的发病机制中具有非常重要的作用，激活该通路可直接参与肝胆管结石病的发生，但具体机制尚不明确。因此，进一步探索LPS/TLR4信号通路在肝胆管结石病发病机制中的激活过程与作用至关重要。这将对肝胆管结石发病机制提供新的见解，同时为研究治疗及干预肝胆管结石病的措施指明新方向。

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