药学学报

胆汁酸转运体与相关疾病及药物研发进展

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段晓延 , 扈金萍 ^*

药学学报 | 综述 2022,57(12): 3576-3586

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药学学报 | 综述 2022, 57(12): 3576-3586

胆汁酸转运体与相关疾病及药物研发进展

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段晓延, 扈金萍^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 创新药物非临床药物代谢及PK/PD研究北京市重点实验室, 北京 100050

通讯作者:

*扈金萍, Tel: 86-10-50927172, E-mail: hujp@imm.ac.cn

Recent advances in drug development targeting bile acids transporters and related disease

Xiao-yan DUAN, Jin-ping HU^*

Affiliations

Beijing Key laboratory of Non-Clinical Drug Metabolism and PK/PD Study, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2022-12-12 doi: 10.16438/j.0513-4870.2022-0889

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摘要

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胆汁酸(bile acids, BAs) 是胆汁的主要成分之一, 在人体的脂肪代谢过程中起到重要作用。人体中95%的胆汁酸中通过肠肝循环而维持, 在肠肝循环过程中, 存在4个主要介导胆汁酸进行跨膜转运的转运体, 每一个转运体都发挥着不可或缺的作用。已有病例报道, 胆汁酸转运体的基因缺陷会导致肠肝循环被破坏, 引发严重的代谢性疾病, 甚至威胁到患者生存。另一方面, 代谢性疾病状态下的患者胆汁酸转运体表达也会发生改变, 这提示胆汁酸转运体的改变或许是某些代谢性疾病发生的重要病理机制。因此, 以胆汁酸转运体为靶点的新药研发正在成为研究热点。本综述探讨了不同胆汁酸转运体与疾病发生的关系, 并对胆汁酸转运体靶点药物研发的最新进展进行总结, 为后续针对胆汁酸转运体的研究提供指导。

关键词

胆汁酸 / 肠肝循环 / 牛磺胆酸钠协同转运多肽 / 胆盐输出泵 / 顶端钠依赖性胆汁酸转运体 / 有机溶质转运体α/β

Abstract

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Bile acids (BAs) are a major component of bile salt, which plays a vital role in the metabolism of lipids in humans. Ninety-five percent of bile acids are recycled by the enterohepatic circulation (EHC), and therefore EHC is essential for bile acid homeostasis. There are four transporters that mediate the transmembrane transport of bile acids, each of which plays an important role in the enterohepatic circulation. Gene defects in bile acid transporters can lead to disorders of the enterohepatic circulation, ultimately leading to clinical phenotypes such as metabolic diseases and even death. Bile transporter expression is altered in patients with various metabolic disease states, suggesting that disruption of bile acid transporters may be a pivotal pathological mechanism for the development of metabolism diseases. Thus, many drugs targeting bile acid transporters are being developed. We provide a concise overview of the progress of bile acid transporters research, discuss the relationship between different bile acid transporters and disease development, and summarize the current progress in drug development targeting bile acid transporters.

Key words

bile acid / enterohepatic circulation / sodium taurocholate cotransporting polypeptide / bile salt export pump / apical sodium-dependent bile acid transporter / organic solute transporter α/β

引用本文

段晓延, 扈金萍. 胆汁酸转运体与相关疾病及药物研发进展. 药学学报, 2022 , 57 (12) : 3576 -3586 . DOI: 10.16438/j.0513-4870.2022-0889

Xiao-yan DUAN, Jin-ping HU. Recent advances in drug development targeting bile acids transporters and related disease[J]. Acta Pharmaceutica Sinica, 2022 , 57 (12) : 3576 -3586 . DOI: 10.16438/j.0513-4870.2022-0889

正文

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1 胆汁酸的种类、合成代谢、生理功能与调控

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胆汁酸按其结构可分为两大类: 一类为游离型胆汁酸(free bile acid), 包括胆酸(cholic acid, CA)、脱氧胆酸(deoxycholic acid, DCA)、鹅脱氧胆酸(chenodeoxycholic acid, CDCA) 和少量的石胆酸(lithochalic acid, LCA); 另一类为结合型胆汁酸(conjugated bile acid), 主要包括甘氨胆酸(glycocholic acid, GCA)、甘氨鹅脱氧胆酸(glycochenodeoxycholic acid, GCDCA)、牛磺胆酸(taurocholic acid, TCA) 及牛磺鹅脱氧胆酸(taurochenodeoxycholic acid, TCDCA), 是游离型胆汁酸与甘氨酸或牛磺酸结合的产物。

胆汁酸是以肝中央静脉周围的肝细胞中的胆固醇为原料, 在约15种酶的作用下合成的两性类固醇分子^[1], 其合成有两种途径, 经典途径(也称为中性途径) 通过胆固醇-7α-羟化酶(cytochrome P450 family 7 subfamily A member 1, CYP7A1) 启动, 再经过类固醇-12α-羟化酶(cytochrome P450 family 8 subfamily B member 1, CYP8B1) 介导的12α羟化、类固醇-27-羟化酶(cytochrome P450 family 27 subfamily A member 1, CYP27A1) 介导的27α羟化、侧链断裂等步骤, 最终生成CA与CDCA; 替代途径(也称为酸性途径) 则通过CYP27A1启动, 最终生成CDCA。经典途径占总胆汁酸合成的75%。由于CA和CDCA由胆固醇直接合成, 故又称为初级胆汁酸(primary bile acids), 初级胆汁酸在肠道内经肠道菌群作用进行7α脱羟作用生成次级胆汁酸(secondary bile acids)^[2], 包括DCA、LCA等。大多数LCA与粪便一起排泄, 少量LCA经过肠肝循环到肝脏并通过硫酸化迅速结合并排泄到胆汁中。

胆汁酸分子含有亲水性的羟基、羧基或磺酸基, 也含有疏水性的烃链, 赋予胆汁酸分子两亲性。亲水性基团与疏水性基团分别位于胆汁酸分子环戊烷多氢菲核的两侧, 从而使胆汁酸分子具有表面活性, 能够插进油相与水相之间, 降低油/水两相的表面张力, 促进脂类乳化, 进而增加其与脂肪酶的接触面积, 加速脂肪的消化。此外, 胆汁酸可与卵磷脂、胆固醇组成混合胶束, 防止胆固醇在胆汁中过饱和而形成胆石沉淀, 再加之胆汁酸的合成是胆固醇重要的消除途径, 因此, 胆汁酸对于机体的胆固醇稳态至关重要^[3]。

胆汁酸的合成受到自身的负反馈调节。研究表明, 胆汁酸合成的负反馈调节主要与胆汁酸合成的限速酶CYP71A基因转录受到抑制有关^[4]。胆汁酸被合成后, 与肝脏上的法尼酯X受体(farnesoid X receptor, FXR) 结合, FXR诱导小分子异源二聚体伴侣(small heterodimer partner, SHP) 的生成, SHP作为一种辅助抑制剂抑制了肝相关同源物1 (liver-related homolog-1, LRH-1) 或肝细胞核因子4α (hepatocyte nuclear factor 4α, HNF4α) 的转录活性, 而后两者可与CYP7A1基因启动子结合, 这个过程最终导致CYP7A1基因的转录受到抑制^[4]。也有观点认为, 胆汁酸在进入小肠后, 与小肠的FXR受体结合可激活成纤维细胞生长因子19 (fibroblast growth factor 19, FGF19), FGF19可与成纤维细胞生长因子受体4 (fibroblast growth factor receptor 4, FGFR4) 结合, 从而抑制CYP7A1和胆汁酸的合成。有实验证明, FGF19可通过c-Jun N端激酶(c-Jun N-terminal kinase, JNK) 依赖性通路直接抑制CYP7A1的表达^[5]。FGF19对CYP7A1的抑制在很大程度上取决于细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK) 信号的激活, 但下游目标仍不明确^[6]。已有动物实验证明, FGF15 (小鼠中的FGF19同源生长因子)、FGFR4可与SHP协同调节胆汁酸的合成^[7]。

2 胆汁酸的肠肝循环及其相关转运体

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人体肠道内的食物消化每天需要12~32 g胆汁酸, 而人体肝脏每天仅需合成0.4~0.6 g的胆汁酸便可满足脂类食物消化需求, 这是因为95%的胆汁酸都会经过肠肝循环而被回收利用。肝细胞合成的胆汁酸通过小管膜分泌到胆汁中并储存在胆囊中, 当人体进食后, 胆囊收缩, 将胆汁酸释放到肠道内。在肠道中, 胆汁酸可促进脂质与脂溶性维生素的消化与吸收, 并在回肠中被有效吸收, 通过门静脉血运回肝脏再分泌到胆汁中。这个过程称为胆汁酸的肠肝循环(enterohepatic circulation, EHC)^[8]。只有5%的总胆汁酸(约每天0.5 g) 被排泄到粪便中, 并且通过肝脏合成来补充。这个过程每天重复4~10次。

胆汁酸的相关转运体广泛存在于人体小肠上皮细胞、肝细胞与胆小管上皮细胞膜上, 其中几个主要的转运体被确定为EHC中的驱动因素, 胆盐输出泵(bile salt export pump, BSEP) 以胆汁形式将胆汁酸从肝细胞排出到胆管, 最终到达肠腔; 顶端钠依赖性胆汁酸转运体(apical sodium-dependent bile acid transporter, ASBT) 将胆汁酸从肠腔重吸收到肠细胞中; 有机溶质转运体α/β (organic solute transporter α/β, OSTα/β) 将胆汁酸从肠细胞转运到血液循环; 牛磺胆酸钠协同转运多肽(sodium taurocholate cotransporting polypeptide, NTCP) 将胆汁酸从门静脉吸收到肝细胞中, 这4个转运体连接了EHC的整体过程, 如图 1。此外, 还有其他几种转运蛋白或酶也参与到EHC的过程中。在病理情况下, 以胆汁酸相关的转运体为靶点可能会成为新的药物研发思路^[9]。

3 肝脏相关的胆汁酸转运体

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3.1 肝脏基底外侧摄取转运体: 牛磺胆酸钠协同转运多肽(NTCP)

3.1.1 NTCP分布与转运特性

NTCP主要分布在肝脏细胞的基底膜上, 由位于人14号染色体上的SLC10A1基因编码, 是一个协同转运体, 它利用钠的浓度梯度将门静脉中的胆汁酸(特别是结合型牛磺胆汁酸) 转运进入肝脏细胞, 参与胆汁酸的肠肝循环过程。除胆汁外, NTCP还可以转运类固醇激素和多种药物, 最近的研究表明, NTCP很可能也参与脂肪酸和甘油磷脂的代谢, 以及色氨酸、泛酸和辅酶A的生物合成^[10]。NTCP是介导肝细胞吸收胆汁酸的主要转运体, 体外实验早已证明, NTCP的下调降低了牛磺胆汁酸的转运^[11]。而在以往NTCP-KO小鼠中表现为高胆固醇血症, 提示肝细胞胆汁酸摄取降低, 但未表现出明显的肝损伤或其他解剖学异常^[12]。

3.1.2 NTCP与疾病发生

已有报道证明, 不同种族的人群中NTCP编码区存在7个单核苷酸多态性, 导致了体外转运功能的降低, 但是与其他转运体不同, NTCP的多态性发生较为罕见, 在370个受试者中仅发现了4个非同义单核苷酸多态性^[13]。临床上NTCP缺陷的病例在近年被相继报道, 2015年Vaz等^[14]报道了第一例NTCP缺陷病例, 表现为高胆固醇血症与正常水平的胆红素, 发育不良但无瘙痒或黄疸。后续相继报道的病例显示, NTCP缺乏的发生大多是一个良性的过程, 大多数患者是无症状的, 唯一显著的差异是血清胆汁酸峰值水平, 这可能是由于肝细胞上其他转运体(例如有机阴离子转运多肽) 摄取结合型胆汁酸的增加以及胆汁酸重新合成的下调有关^[15]。并且, 随时间的增加, NTCP缺陷患者体内胆汁酸水平的硫酸化与胆汁酸水平的正常化呈正相关, 这提示胆汁酸的硫酸化是NTCP缺陷个体的胆汁酸解毒和消除的主要机制^[16]。

3.1.3 靶向NTCP的药物研发

NTCP与肝炎的发生密切相关。近年来的研究显示, NTCP是乙型肝炎病毒(hepatitis B virus, HBV) 及其卫星病毒丁型肝炎病毒(hepatitis D virus, HDV) 的特异性受体^[17], 并且胆汁酸的转运与HBV/HDV的转运可能拥有同一个NTCP结合位点并相互竞争结合^[18]。由于目前丁型肝炎的治疗(例如IFN-α疗法) 有限, 通过抑制NTCP而阻止HBV/HDV进入肝细胞成为慢性丁型肝炎的药物研发新思路^[19]。在2020年, 欧盟通过了HBV/HDV进入抑制剂bulevirtide (myrcludex B, MYR Pharmaceuticals公司开发) 的条件上市, 是全球第一个丁肝治疗药。近期的myrcludex B临床试验中发现, 该药与IFN-α合用治疗丁肝效果显著, 并且耐受性良好, 无不良反应的发生^[20]。

在治疗肝纤维化的药物开发中, Salhab等^[21]最新研究显示, 肝星状细胞(hepatic stellate cells, HSCs) 上NTCP的表达与肝纤维化的严重程度呈正相关, NTCP介导了HSCs对胆汁酸的摄取, 促进HSCs激活, 活化的HSCs表现出促纤维化的转录和分泌特性, 是肝纤维化发生发展过程中的重要环节。在肝纤维化的小鼠中, 通过靶向抑制NTCP可以减少胆汁酸在肝脏中的积累, 从而缓解肝纤维化进程。此外, 通过靶向抑制NTCP, 也可以对小鼠饮食诱导的肥胖与肝脏脂肪变性起到保护作用^[21-23], 这可能是因为胆汁酸摄取减少导致外周胆汁酸的上升, 激活FXR受体, 诱导tgr5基因的表达, 刺激胰高血糖素肽-1的分泌, 进而改善肝脏葡萄糖和脂质的代谢^{[24, 25]}。因此, 靶向NTCP介导的胆汁酸摄取可能会成为治疗肥胖和肥胖相关的肝硬化的一种新方式。胆囊异常、胆汁淤积症等疾病也已被证实与NTCP有密切关系。Mao等^[26]对NTCP-KO小鼠进行病理检查发现胆囊异常表型(包括胆囊壁增厚、上皮细胞异常增殖与粒细胞浸润等), 并在临床研究中也发现了NTCP缺乏与胆囊异常的相关性。Slijepcevic等^[27]使用myrcludex B减少肝脏的胆汁酸负荷, 改变胆汁中磷脂与胆盐的比率来减轻小鼠的胆汁淤积性肝损伤。以上数据表明, NTCP可成为代谢性肝病、肝纤维化等疾病治疗的新靶点。由于NTCP与FXR的反向调节作用^[21], 通过抑制NTCP来增强FXR功能或许可以替代FXR激动剂来治疗代谢性肝病, 并减缓FXR激动剂的不良反应, 例如高胆固醇血症和瘙痒^[28]。

3.1.4 其他的肝脏基底外侧摄取转运体

除NTCP外, 有机阴离子转运多肽(organic anion transporting polypeptides, OATPs) 可介导非钠离子依赖性胆汁酸的摄取, 所有OATP具有广泛的底物选择性, 可转运游离胆汁酸、结合胆汁酸、胆红素和许多药物^[9]。人与啮齿类动物肝细胞表达的OATP的亚型不同, 小鼠肝脏主要表达OATP1A1、-1A4、-1B2和-2B1, 而人类肝脏主要表达OATP1B1和OATP1B3^[29]。人类OATP多态性影响内源性胆汁酸摄取的证据很少^[9]。OATP是肝脏摄取游离型胆汁酸的主要转运体, 但对结合型胆汁酸的摄取却具有种属差异。Slijepcevic等^[12]研究显示, NTCP-KO的小鼠模型中一些小鼠表现出正常的结合型胆汁酸水平, 可能由于OATPs的表达发生改变, 建立了胆汁酸的代偿性清除机制, 提示OATPs在小鼠肝脏对结合型胆汁酸的摄取同样重要。随后研究显示, OATPs-KO小鼠在Myrclucdex B处理后血清结合型胆汁酸水平升高, 这提示NTCP和OATPs介导小鼠肝脏对结合胆汁酸的摄取, 而人肝脏主要由NTCP介导摄取^[30]。

此外, 早期研究显示, 微粒体环氧化物水解酶(microsomal epoxide hydrolase, mEH) 也在肝脏细胞基底外侧对胆汁酸摄取中发挥作用^[31], 然而最近研究发现, mEH并不介导结合型胆汁酸的转运^[30], mEH介导胆汁酸转运的体外证据也未独立验证^[32], 因此关于mEH对胆汁酸的转运还需要进一步研究。

3.2 肝脏顶端外排转运体: 胆盐输出泵(BSEP)

3.2.1 BSEP的分布与转运特征

BSEP是一个ATP-结合盒(ABC家族) 转运体, 主要负责胆汁酸的转运。BSEP主要表达在肝脏小管膜上。在肝外其他组织中, 睾丸、气管、前列腺、肺、胸腺、肾脏和结肠等组织中也可检测到一定水平的BSEP mRNA, 但仅在肝细胞中检测到功能蛋白质的顶膜表达^[33]。早在1995年, BESP作为P-糖蛋白的姐妹转运体被发现^[34], 其具有两个跨膜结构域和两个核苷酸结合结构域的特征。BSEP主要以ATP依赖的方式转运结合胆汁酸(包括GCA、GCDCA、TCA、TCDCA)^[35], 却不转运CA等非结合胆汁酸^[36]。此外, BSEP对底物胆汁盐的特异性较小, 也可转运普伐他汀、长春碱等药物。

3.2.2 BSEP与疾病发生

BSEP由ABCB11基因编码, 早在1998年就有文献^[37]发现了患者的BSEP突变缺陷, 经过大量研究发现, BSEP的ABCB11基因突变与多种肝细胞源性胆汁淤积疾病有关, BSEP转运蛋白的功能障碍导致进行性家族性2型肝内胆汁淤积(progressive familial intrahepatic cholestasis type 2, PFIC2)、良性复发性2型肝内胆汁淤积和妊娠肝内胆汁淤积^[33]。PFIC2是一种以慢性肝内胆汁淤积为特征的临床综合征, 通常开始于婴儿期, 并在10~20年间发展为胆汁性肝硬化和肝功能衰竭^[38]。患者常表现为高血清胆汁酸浓度、高胆红素血症、低或正常的血浆γ-谷氨酰转移酶水平。BSEP缺陷型胆汁淤积被认为与BSEP顶膜定位缺失与胞浆滞留有关。有研究发现, PFIC2的一些错义突变(G238V、E297G、G982R、R1153C和R1268Q) 可以导致BSEP在顶膜的插入受损, 这可能因为突变引起蛋白折叠错误, 膜蛋白被隔离在胞质中, 随后被泛素/蛋白酶体系降解^[39]。BSEP转运蛋白从高尔基体合成后, BSEP先在顶膜下区室(subapical compartment, SAC) 停留, 并受到Rab11蛋白的调控, 随后在SAC和顶膜之间形成动态运输循环^[40]。Wu等^[41]研究表明, 荷电多囊体蛋白5 (charged multivesicular body protein 5, CHMP5) 介导了BSEP在SAC的定位。BSEP在CHMP5作用区突变会导致BSEP插入顶膜受损并潴留在SAC中。与人类相比, 小鼠BSEP缺乏表现出的胆汁淤积相对温和, 具有较高的生存活性, 胆汁酸分泌减少但总胆汁酸分泌仍占据野生型小鼠的30%, 这表明小鼠拥有另一套胆汁酸转运系统, 以弥补BSEP的缺失^[42]。随后研究证实, 多药耐药(multidrug resistance, MDR) 的MDR1和MDR2在BESP敲除小鼠中过量表达^[43], 并介导胆汁酸的转运, 而这可能就是人与小鼠BESP缺陷表型差异的原因。

3.2.3 靶向BSEP的药物研发

针对BESP功能障碍性疾病的药物主要以增强转运体功能和表达为主。UDCA是治疗胆汁淤积最常用的药物, 通过纠正BESP突变体的潜在运输缺陷以及减少转运体的内化来发挥作用^[44]。FXR受体是控制BESP表达的主要配体激活转录因子, 因此通过靶向激活FXR受体来增加BSEP的表达成为新的BSEP缺陷的治疗方向。奥贝胆酸(obeticholic acid, OCA) 是一种人类主要胆汁酸CDCA的衍生物和FXR激动剂, 被FDA批准用于原发性胆管炎(primary biliary cholangitis, PBC) 和非酒精性脂肪性肝炎(non-alcoholic steatohepatitis, NASH)。有研究表明, OCA可以在临床上逆转部分组织病理学特征^[45]。4-苯基丁酸钠(4-phenylbutyric acid, 4-PBA) 是一种协助蛋白质转录后修饰和折叠的分子伴侣, 在体外研究中发现可以增强BSEP的顶膜插入, 增强TCA转运活性^[46], 而临床研究中也发现用4-PBA治疗PFIC2患者可以改善血清胆汁酸浓度、瘙痒评分等疾病标志物^{[47, 48]}。此外, van Wessel等^[49]最新研究发现, BESP无义突变导致框内终止密码子和功能蛋白的缺失, 并增加了发展为肝癌的风险。而有文献^[50]表明, 氨基糖苷类抗生素庆大霉素与核糖体结合, 并在提前终止密码子处诱导翻译, 从而导致全长蛋白合成的部分恢复。Amzal等^[51]最新研究体外评估庆大霉素对6种BSEP无义突变(Y354X、R415X、R470X、R1057X、R1090X和E1302X) 的影响, 所有的通读结果都有显著增加, 这提示着治疗BESP缺陷相关疾病的新方向。

3.2.4 其他的肝脏顶端外排转运体

多药耐药蛋白2 (multidrug resistance-associated protein 2, MRP2) 定位于肝细胞顶端侧与肾脏近端小管的管腔膜上, 介导包括结合型胆汁酸在内的两亲性结合物, 却不转运游离型胆汁酸^[52]。此外, 还原型谷胱甘肽也可通过MRP2转运排泄到胆管中^[53]。MRP2基因缺陷可导致结合型高胆红素血症, 表现为血清胆汁酸升高, 胆汁分泌减少, 这可能与肝细胞内多药耐药蛋白3和多药耐药蛋白4介导的胆汁酸向血液循环外排增加有关^[54]。

4 肠道相关的胆汁酸转运体

收起

4.1 肠壁顶端摄取转运体: 顶端钠依赖性胆汁酸转运体(ASBT)

4.1.1 ASBT分布与转运特性

ASBT主动从肠腔中重吸收胆汁酸, 在EHC中扮演重要的角色, 并对胆汁酸稳态发挥重要作用。ASBT主要分布在回肠末端肠细胞的顶端刷状缘膜上, 在肾脏近端小管顶膜和大胆管上皮也有相对较低的表达^[55]。在人心脏、肺、大脑、十二指肠、睾丸和子宫中检测到ASBT的mRNA, 但未检测到蛋白质^[56]。ASBT利用细胞内负电位与Na⁺浓度梯度对BAs进行继发性主动转运, 并通过Na⁺/K⁺-ATP酶维持细胞内外电位差与浓度梯度。ASBT的底物谱狭窄, 通常只转运胆汁酸(包括游离型胆汁酸和结合型胆汁酸)。然而ASBT对于不同胆汁酸的亲和力是不同的, 与疏水的游离胆汁酸相比, 亲水性更强的结合型胆汁酸更容易被ASBT所转运^[57], 然而, 二羟基胆汁酸(CDCA和DCA) 对ASBT的亲和力高于三羟基胆汁酸(CA和TCA)。在多种肠道和肝脏胆汁酸转运体中, 回肠ASBT在EHC中是不可替代的, 而OSTα/β、NTCP、BSEP可分别由MRP3、OATPs和MRP1、MDR2和MRP2进行补偿, 即使其底物特异性发生了变化^[57]。此外, 回肠胆汁酸结合蛋白(ileal bile acid binding protein, iBABP) 协助将胆汁酸从肠上皮的顶膜转运到基底外侧膜, 以降低胆汁酸对肠上皮细胞的毒性^[58]。有报道称, ASBT的表达受到肝细胞核因子1-α (hepatocyte nuclear factor-1-α, HNF1-α)^[59]、甾醇反应元件结合蛋白(sterol response element binding proteins, SREBPs)^[60]、尾型同源盒蛋白(caudal type homeobox proteins, CDX)^[61]、FGF15/19与β-Klotho^[62]等蛋白调节因子的调节, 此外, 生理条件下, 肠道菌群也可以通过Gata4抑制小鼠回肠末端的ASBT表达^[63]。一些核受体对ASBT的表达同样起到调控作用, 例如: RAR/RXR顺式反应元件可以激活ASBT启动子^[64], 过氧化物酶体增殖物激活受体α (peroxisome proliferator-activated receptor α, PPAR-α) 也可以增加ASBT启动子活性^[65]。

4.1.2 ASBT与疾病发生

ASBT由SLC10A2基因编码, 与NTCP (SLC10A1编码) 相关, 人类ASBT基因位于染色体13q33上, 为长度约22.8 kb的DNA, 由6个编码外显子组成, 编码一个分子质量为38 kDa的348个氨基酸蛋白质。ASBT缺陷患者早在1997年被发现^[66], 表现为原发性胆汁酸吸收障碍, 人ASBT遗传突变可能导致胆汁酸吸收不良、EHC破坏和血浆胆固醇降低^[58], 也有文献^[9]表明, ASBT缺陷可以导致炎症性肠病、便秘、Alagille综合征、家族性高甘油三酯血症、先天性慢性腹泻、肠易激综合征、NASH等。所以, ASBT自然产生的遗传多态性被认为与胃肠道的各种疾病有关。已知的ASBT产生的多态性与野生型相比, 在蛋白质水平表达或膜表面运输方面没有显著差异, 因此ASBT的遗传多态性与胃肠道疾病有关可能是因为ASBT转运活性下降^[67]。

4.1.3 靶向ASBT的药物研发

虽然ASBT缺陷可导致严重的临床表型, 但是靶向抑制ASBT依然可以作为调节肠腔、肠细胞、肝细胞和体循环中胆汁酸含量的方法, 这主要是通过减少胆汁酸重吸收, 负反馈增加胆汁酸的合成并降低胆固醇含量, 从而对多种肠道和代谢性疾病产生治疗效果。

胆汁酸可与肠细胞膜上TGR5 (G protein-coupled bile acid receptor 1) 结合, 刺激环磷酸腺苷(cyclic adenosine monophosphate, cAMP) 和氯化物的产生, 导致结肠电解质分泌增加。因此, ASBT抑制剂通过减少胆汁酸重吸收而增加肠道内胆汁酸含量, 进而缓解便秘等相关症状^[68]。Elobixibat (A3309, 由Albireo公司开发的ASBT抑制剂) 已被研究用于治疗慢性先天性便秘(chronic constipation, CC) 和便秘型肠易激综合征(irritable bowel syndrome, IBS), elobixibat可使患者自发排便次数增加, 结肠传输时间缩短, 导致粪便稠度可逆性松动^[69]。在日本的Ⅲ期临床研究中, 日剂量5~15 mg的elobixibat疗效较好, 不良反应为腹痛与腹泻, 长期试验中耐受性较好^{[70, 71]}。与其他治疗CC的药物相比, elobixibat具有增加结肠蠕动与增加结肠分泌双重作用^[56]。Elobixibat于2018年在日本上市, 并有望在将来在欧洲与美国上市。

由于异常胆固醇代谢在肝脏中积累是NASH发病的促进因素, 通过抑制ASBT减少体内胆固醇含量是治疗NASH的有效治疗方式。ASBT的抑制一方面可影响回肠内胆汁酸组成, 使得具有FXR激活作用的胆汁酸减少(例如CDCA), 引起回肠FXR的抑制与FXR介导的FGF15/19释放减少, CYP7A1活性增强, 更多的胆固醇被催化为胆汁酸, 从而达到消耗胆固醇的目的^[72]。另一方面, ASBT的抑制也改变了肝脏胆汁酸组成, 使得具有FXR激活作用的胆汁酸增多, 肝脏FXR激活, 胆汁酸合成增加并减少脂质合成基因Srebp1的表达, 从而减少胆固醇堆积^[73]。此外, 胆固醇储备的减少进一步降低了血浆低密度脂蛋白(low density lipoprotein, LDL) 水平, 这使得靶向ASBT而治疗血脂紊乱成为可能。Elobixibat的早期临床研究中观察到C4 (胆固醇代谢和胆汁酸合成的生物标志物) 浓度依赖性增加与总胆固醇、LDL的降低^[69], Rudling等^[74]则发现elobixibat使血脂异常患者LDL/HDL比值降低了18%。高脂饮食诱导的NASH小鼠模型中, 使用ASBT抑制剂被证实可以增加血浆C4并阻止纤维化进展。然而, 2020年elobixibat的II期临床试验中发现, elobixibat虽然在降低LDL方面疗效显著, 但是在降低谷丙转氨酶和改善肝脏脂肪含量方面(NASH更为重要的治疗指标), 疗效与安慰剂组无显著性差异, 因此elobixibat最终因疗效差而停止在治疗NASH上的开发。同年, volixibat (LUM002, Shire公司开发的另一个ASBT抑制剂) 的Ⅱ期临床试验也因为疗效较差而终止, 这提示NASH发病具有复杂性, 针对单一转运体并不一定能获得较好疗效^[75]。而Matye等^[76]对NASH小鼠使用ASBT抑制剂联合FGF15类似物进行治疗, 结果表明两种药物联用疗效优于任意一种药物的单独使用的疗效, 此外联合应用还可以减轻两种药物分别的临床不良反应——腹泻和高胆固醇血症, 因此, 与其他药物联用或许可以为ASBT抑制剂的开发提供新的思路。

ASBT治疗胆汁淤积主要是通过减少胆汁酸重吸收来降低肝脏的胆汁酸负荷^[77]。而CYP7A1被诱导导致的胆汁酸合成增加也不能抵消胆汁酸的受损^[78]。此外, ASBT抑制还改变了胆汁酸组成, 使得具有细胞毒性疏水性胆汁酸减少。临床研究中, 用ASBT抑制剂A4250对胆汁淤积患者治疗中发现, A4250对胆汁淤积患者的瘙痒症状有明显改善, 但出现腹痛、腹泻等不良反应, 可能与肠蠕动增加有关^[79]。

Wu等^[80]发现, 在大鼠模型中胆汁酸肠道转运的减少将通过影响胰高血糖素样肽-1 (glucagon-like peptide-1, GLP-1) 的分泌影响葡萄糖稳态, 导致胰岛素水平升高, 糖化血红蛋白和血糖下降, 这提示ASBT抑制剂也有用于治疗2型糖尿病(diabetes mellitus type 2, T2DM) 的可能。在上述的elobixibat的临床研究中也观察到血液循环中GLP-1的峰值浓度增加^[74]。目前, 进入T2DM治疗临床研究的ASBT抑制剂主要是linerixibat (GSK-2330672)。Linerxibat在Ⅱ期临床试验中与二甲双胍联用, 在降低患者血糖、血脂方面疗效优于考来维伦(胆汁酸螯合剂)^[81]。与其他治疗T2DM的药物相比, ASBT抑制剂具有同时控制高脂血症与高血糖的优势。

4.2 肠壁基底侧外排转运体: 有机溶质转运体α/β (OSTα/β)

4.2.1 OSTα/β的分布与转运特性

OSTα/β是一种异聚溶质载体蛋白, 由Ballatori团队^[82]于2001年首次发现, 由两个非同源亚基共表达形成了功能性胆汁酸转运体。在肾上腺皮质网状带、肾小管和小肠等肠组织中的上皮细胞基底膜外侧均检测到OSTα/β, 而在回肠和十二指肠中发现的OSTα/β的mRNA与蛋白质水平最高^[83]。在同一组织中, OSTα与OSTβ的蛋白表达量也是不同的, 有趣的是, 在OST生理功能明显的肠细胞中, 或是胆汁淤积的肝细胞中, OSTβ的蛋白表达量高于OSTα, 而在OSTα/β作用尚不清楚的组织中, OSTα的蛋白表达量最高^[84]。OSTα/β与位于肠细胞顶端膜上的ASBT协同, 介导了胆汁酸从肠道进入血液循环的过程。此外, OSTα/β介导的胆汁酸外流也保护了小肠上皮细胞免受细胞内胆汁酸积累和肠道损伤^[85]。对于不同类型的胆汁酸, OSTα/β也具有不同的亲和力。对OSTα/β过表达细胞的动力学研究表明, 与游离型胆汁酸相比, 结合了甘氨酸与牛磺酸的结合型胆汁酸与OSTα/β的亲和力明显增加^[86]。而在结合型胆汁酸中, OSTα/β优先运输胆汁酸的顺序如下: TCDCA > GCDCA > TCA > GCA。这说明, 与亲水性的TCA与GCA相比, OSTα/β优先转运亲脂性的TCDCA和GCDCA, 而与甘氨酸官能团相比, OSTα/β优先转运具有牛磺胆酸官能团的结合型胆汁酸^[87]。除了胆汁酸, 一些类固醇物质与外源性药物也可作为OSTα/β的底物被转运。硫酸雌酮(estrone sulfate, ES)、前列腺素E2被证实可在非洲爪蟾卵母细胞中被OSTα/β转运^[82], 硫化孕烯雌酮也被证实为OSTα/β的底物^[88]。在药物底物中, OSTα/β与P-gp、OATP1B3最为相似^[84], 可转运地高辛、他汀类药物等。地高辛被证实可在非洲爪蟾卵母细胞中被OSTα/β所转运^[82], 而Li等^[89]通过Caco-2细胞与OSTα/β抑制剂合用推测OSTα/β可能介导了阿托伐他汀从基底侧向顶端侧转运的可能。

4.2.2 OSTα/β与疾病发生

OSTα和OSTβ分别由两个位于不同染色体上的基因编码(分别为位于Chr3和Chr15的SLC51A和SLC51B)。在哺乳动物细胞中, OSTα和OSTβ相结合形成异型的OSTα/β复合物, 并且, 与单独表达一个OST亚基的细胞相比, OSTα和OSTβ的共表达的细胞可以显著提高其对TCA和ES的摄取。OSTα/β是胆汁酸的肠肝循环的重要组成部分, 其基因的缺陷会导致肠细胞外排胆汁酸的受限, 最终破坏胆汁酸稳态。Rao和Ballatori等^{[90, 91]}通过对小鼠SLC51A基因的敲除, 建立OSTα-KO小鼠模型, 小鼠表现为肠道胆汁酸重吸收大幅减少, 并且胆汁酸池大量减少, 这与肠道胆汁酸减少导致FXR强烈激活有关^[92]。肠道表型为小肠绒毛减少, 肠壁增厚, 并且随着ASBT基因的敲除而恢复, 这表明OSTα/β在保护肠道上皮细胞免受细胞内胆汁酸累积损害而发挥重要作用^[85]。人类的OSTα/β基因缺陷在近年才被发现。2018年在两患者中发现了SLC51B的基因缺陷型^[93], 临床特征为慢性腹泻、严重脂溶性维生素缺乏和胆汁淤积性肝病的特征, 包括肝酶升高, 特别是血清中γ-谷氨酰基转移酶活性升高, 肝活检显示轻度的门静脉纤维化。直到最近才发现一名SLC51A基因缺陷的患者^[94], 肝活检显示肝小叶结构伴门静脉周围纤维化, 提示早期肝硬化和轻微炎症。与SLC51B基因缺乏相比, 其临床表现更为严重, 容易出现瘀伤, 两次持续性出血(可归因于脂溶性维生素K吸收不良), 并最终以生存失败告终。OSTα和OSTβ缺陷似乎有一个共同点, 即患者表现为慢性吸收不良性腹泻, 但是由于患者数量较少, 很难有更深入的了解。

4.2.3 靶向OSTα/β的药物研发

已有体外与体内实验证明, 胆汁淤积状态可以影响OST的蛋白表达。Boyer等^[95]用CDCA处理HepG2细胞系, 显著提高了OSTα、OSTβ和BSEP mRNA的含量, 并增加OSTα和OSTβ的蛋白表达。而在人体样本中发现, 肝外胆汁淤积的患者的OSTα/β被诱导表达增加^[96], 这提示毒性胆汁酸成分在肝脏积累时, OSTα/β介导的外排具有重要的解毒作用。有趣的是, OSTα/β在正常人体肝脏中表达很低, 但是却在PBC等胆汁淤积患者肝脏中高度表达(特别是OSTβ蛋白高度表达)^[95]。Chai等^[97]研究表明, 在胆汁淤积患者中, SLC51B的mRNA含量和OSTβ蛋白表达显著提高, 而SLC51A含量保持不变, OSTα的蛋白表达却降低。而在NASH患者中除了发现SLC51B mRNA和OSTβ蛋白的显著增加, OSTα的蛋白表达也有所提高^[98]。肝脏OSTα/β在胆汁淤积状态的表达增加, 意味着疾病状态可能会增加经OSTα/β介导转运的药物产生毒性的风险。

虽然目前还并未存在一个上市药物以OSTα/β靶点进行治疗, 但是针对OSTα/β为靶点进行药物设计却存在理论基础。利用FXR激动剂OCA治疗PBC可以显著诱导三明治培养人肝细胞中OSTα/β表达, 从而增加TCA的外排, 减少其在细胞中的积聚与毒性作用^[99], 但是目前尚不清楚OSTα/β的介导的胆汁酸外排增强在OCA治疗PBC中发挥多大作用。Zollner等^[100]通过建立FXR-KO小鼠模型证实胆汁酸喂养诱导的OSTα/β表达增加是通过FXR通路介导的, 并且推测在胆汁淤积中治疗性诱导OSTα/β表达可以保护肝脏免受肝毒性, 这似乎是一个很合理的肝脏保护机制。因此, 针对OSTα/β为靶点进行药物研发具有一定前景。

4.2.4 其他的肠壁基底侧外排转运体

介导肠细胞内胆汁酸外排的另一个转运体是多药耐药蛋白3 (multidrug resistance-associated protein 3, MRP3), MRP3在大鼠和人类小肠中被发现^{[101, 102]}, 在所有肠段的基底外侧膜均有表达, 但在十二指肠中的表达量最低, 而在末端回肠和结肠中表达显著增加。与野生型小鼠相比, MRP3-KO小鼠表现为肝胆汁酸含量增加, 而血清胆汁酸含量则下降^[103]。然而在胆管结扎后, MRP-KO小鼠和野生型小鼠之间的组织学肝损伤和血清胆汁酸水平并没有显著性差异^{[104, 105]}, 这表明MRP3对于胆汁酸的肠吸收可能不是必需的。

5 总结

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随着分子生物学发展与结构生物学等新兴学科的兴起, 人们对于胆汁酸转运体的结构功能、分子调控机制及与代谢性疾病发生发展的关系有了全新的认识。目前, ASBT、BSEP和NTCP的冷冻电镜结构相继被解析^[106-109]。这些结构解析都会对于将来转运体底物、抑制剂鉴定, 靶向转运体药物的筛选有很大帮助。然而针对胆汁酸转运体的研究仍任重道远, 许多问题仍有待解决, 例如mEH是否对胆汁酸的EHC过程发挥作用？OSTα/β介导的肝细胞保护作用是否是OCA治疗PBC的主要机制？因此, 应通过深入机制研究, 探索药物靶点开发, 为未来治疗提供帮助。

作者贡献: 段晓延负责文献查阅、撰写; 扈金萍负责文献选题、写作指导与修改。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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中国医学科学院医学与健康科技创新工程项目(2022-I2M-2-002)

参考文献

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2022年第57卷第12期

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doi: 10.16438/j.0513-4870.2022-0889

接收时间：2022-07-21
首发时间：2025-12-24
出版时间：2022-12-12

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收稿日期：2022-07-21
修回日期：2022-09-09

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中国医学科学院医学与健康科技创新工程项目(2022-I2M-2-002)

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中国医学科学院、北京协和医学院药物研究所, 创新药物非临床药物代谢及PK/PD研究北京市重点实验室, 北京 100050

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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