胆道梗阻后肠道分泌和吸收功能的改变及其细胞分子机制研究

Biliary obstruction, which is characterized by inducing intestinal bile deficiency and accumulation of biliary compounds in the body, is a common surgical condition and may cause multiple pathophysiologic changes. However, intestinal secretion and absorption function under the condition of biliary obstruction was less studied. It has been discovered that bile acids are regulatory molecules in the previous studies. Bile acids could regulate intestinal physiologic functions via activating specific nuclear receptors (FXR, PXR) and cell signaling pathways (ERK1/2, JNK1/2) to induce the alterations of numerous protein and gene expressions. Our previous studies demonstrated that biliary obstruction could alter intestinal Cl- secretion and glucose absorption. In the present study, we aimed to further analyse the alterations of intestinal Cl- secretion and glucose absorption through using an experimental model of murine biliary ligation and investigate the role of bile acids specific nuclear receptors, cell signaling regulators and cytokines in biliary obstruction-induced intestinal Cl- secretion and glucose absorption alterations through in vitro and in vivo experiments, and the correlation between the expression levels of intestinal mucosal Cl- transport protein, glucose transport protein and clinical features in patients with biliary obstruction. Thus, this study will elucidate the alterations of intestinal Cl- secretion and glucose absorption function after biliary obstruction and its underlying cellular and molecular mechanisms, and may provide a new strategy for preventing biliary obstruction complications.

胆道梗阻后由于肝脏胆汁淤积肝功能受损以及缺少胆汁酸进入肠道这两大主要因素，会导致一系列病理生理改变。胆道梗阻后有关肠道分泌和吸收功能的研究甚少。近10余年的研究表明胆汁酸是一个重要的调节分子，可通过核受体FXR和PXR以及ERK1/2、JNK1/2细胞信号转导通路调控蛋白质和基因表达，进而调节肠道的生理功能。我们前期研究表明胆道梗阻可以导致肠道氯离子分泌和葡萄糖吸收功能的改变。在本项目中，我们将建立小鼠胆道梗阻模型，进一步分析肠道氯离子分泌和糖吸收功能的变化，并通过体内和体外研究阐明胆汁酸特异性核受体、细胞信号调节分子和细胞因子在胆汁酸对肠道氯离子转运及糖吸收的影响中的作用及机制，明确胆道梗阻病人肠粘膜氯离子转运及葡萄糖转运蛋白表达水平的异常及其与临床病理特征的关系，从而深入探讨胆道梗阻后肠道氯离子分泌和葡萄糖吸收功能的变化及其细胞分子机制，有望为胆道梗阻后并发症的防治提供新的理论基础。

胆道梗阻是继发于胆管结石或肿瘤、胆道狭窄或闭锁、以及壶腹部或胰腺头部肿瘤等疾病的临床常见病症，可导致肝脏胆汁淤积和肠道内胆汁酸缺乏。然而，胆道梗阻后肠道吸收和分泌功能的改变及其细胞分子机制，鲜有研究。本项目围绕胆道梗阻及胆汁酸在肠道葡萄糖吸收与氯离子分泌中的作用及其机制开展研究，获得以下结果：（1）胆道梗阻可引起肠道葡萄糖吸收功能和氯离子分泌水平增加，与肠道中葡萄糖转运蛋白SGLT1和氯离子转运蛋白CFTR、NKCC1表达增加有关，而与血液中细胞因子TGF-β和IL-6水平升高无关；（2）胆汁酸可通过肠道葡萄糖转运体SGLT1、GLUT2影响血糖水平，通过氯离子转运蛋白CFTR、NKCC1调控肠道分泌；（3）基因芯片检测胆道梗阻后空肠组织的基因表达，并将差异基因进行聚类、通路分析，发现胆道梗阻后胆汁酸受体FXR及多种与物质代谢、转运相关的基因、通路发生改变；（4）利用siRNA、qRT-PCR、免疫印迹、免疫荧光、免疫共沉淀技术等技术，证实胆汁酸可通过FXR增加小肠细胞GLUT2的表达，进而促进细胞葡萄糖吸收，而这一过程依赖于S1PR2调节的MEK/ERK通路的激活;此外，胆汁酸激活FXR可抑制细胞的氧化磷酸化过程，减少ATP生成，从而影响细胞的葡萄糖转运；（5）胆汁酸激活的FXR可通过调整小肠组织刷状缘膜上GLUT2与SGLT1的比例，抑制肠道中葡萄糖的跨上皮转运，从而降低血糖水平；（6）临床研究表明，胆道梗阻患者肠道中FXR表达降低，易于出现空腹血糖水平升高和腹泻症状，且与肠道内胆汁酸缺乏程度密切相关。此外，皮尔森相关分析显示肠道GLUT2、CFTR mRNA的表达分别与患者血糖水平升高和腹泻症状的出现呈正相关。本研究为胆道梗阻后肠道生理功能的变化以及并发症的防治提供新的理论基础和策略，证实了胆汁酸在调节肠道葡萄糖吸收和氯离子分泌过程中发挥重要作用。同时，胆汁酸及FXR激动剂有望成为治疗胆道梗阻相关性并发症的重要药物。