胆汁淤积下新发现的胆酸排泌泵OATP-D高表达的分子调控机制及其抗肝损伤的效应研究

Chronic cholestasis, caused by many factors, such as bile duct obstruction,inflammation and some drugs, can lead to hepatic fibrosis and liver failure. Adaptive up-regulation of bile acid efflux transporters expression, such as MRP3 and MRP4, are essential to protect the liver from injury in cholestasis. Impaired up-regulation of bile acid efflux transporters can lead to worsen cholestatic liver injury. We first found in our preliminary experiments that a new type of hepatic bile acid efflux transporter, OATP-D is increased dramatically in human obstructive cholestasis. We then demonstrated that OATP-D plays an extremely important role in protecting hepatocytes from injury caused by the intracellular accumulation of toxic bile acids in the preliminary experiments of HepG2 cells and bile duct ligation mice infected with shRNA OATP-D virus. However, the mechanism of functional role of OATP-D gene and specific molecular mechanism of elevated OATP-D gene in obstructive cholestasis are unclear. In our preliminary study, we observed that plasma FGF19 and hepatic FGF19/FGFR4 mRNA were up-regulated in patients with obstructive cholestasis. And ERK/MAPK signaling pathway is also activated in human cholestatic liver. In HepG2 cells, we found that FGF19 activates ERK signaling pathway and stimulates the expression of transcriptional factor SP1 gene. Inhibition of ERK signaling pathway by specific inhibitor PD98059 in HepG2 cells reduces the expression of SP1 gene markedly. Furthermore, SP1 overexpression induces OATP-D expression whereas knockdown of SP1 expression reduces OATP-D expression in HepG2 stable cell lines of SP1 overexpression and SP1 shRNA. All of these results trigger us to formulate a hypothesis: Elevated plasma FGF19 in human obstructive cholestasis could induce transcriptional factor SP1 expression via activating ERK signaling pathway and then up-regulate the expression OATP-D gene. Finally, the dramatic up-regulation of OATP-D gene could excrete the toxic bile acids from cholestatic hepatocytes into blood, protecting the liver from injury. In order to prove this hypothesis, we use different molecular biological techniques, such as OATP-D knockout mice, Real time RT-PCR, western-blot, EMSA, and chip assays, to study the functional role of OATP-D and clarify the molecular mechanism of increased OATP-D gene in human obstructive cholestasis, which will be promising for a clue of the clinical therapy and understanding the cholestasis.

胆汁淤积是多因素所致肝损害的常见并发症，易发展为肝纤维化、肝衰竭。胆汁淤积时，胆酸排泌泵MRP3等表达适应性上调作用关键，其缺失可加重胆汁淤积肝脏的损伤。我们首次发现：新胆酸排泌泵OATP-D在胆汁淤积肝脏特异性高表达，经细胞和小鼠预实验提示它可能有护肝作用。但目前关于OATP-D功能及调控机制研究极少。前期我们发现：胆汁淤积肝脏FGF19/FGFR4表达上调和ERK通路被激活；FGF19可激活HepG2 ERK通路和上调SP1表达，而ERK抑制剂可降低SP1表达；HepG2中SP1过表达和干扰实验表明SP1可调控OATP-D表达。故我们假设：胆汁淤积时FGF19激活ERK通路上调SP1而促进OATP-D高表达，以增强肝细胞抵御胆酸的毒性损伤。本课题拟运用基因敲除、Chip等技术，明确胆汁淤积下OATP-D的护肝作用并阐释其高表达的调控机制。这将为胆汁淤积临床治疗提供新的治疗靶点和理论依据

胆汁淤积下肝细胞膜上胆酸排泌蛋白适应性上调是对抗胆汁淤积的关键因素。我们首次发现OATP3A1/OATP-D/SLCO3A1在胆汁淤积肝脏特异性高表达，但其功能、表达情况及调控机制均不清楚。为此，我们开展了以下研究，并取得相应重要的结果：.1.明确OATP3A1在肝脏的表达分布情况。. 收集人及小鼠胆汁淤积肝组织样本，研究发现OATP3A1在胆汁淤积时高表达；免疫荧光定位发现OATP3A1主要表达于肝细胞基底侧膜。.2. 通过OATP3A1基因敲除小鼠确认其表达上调具有对抗胆汁淤积的效应。. 通过构建了OATP3A1基因敲除小鼠，并建立小鼠胆汁淤积后发现：OATP3A1功能缺失会加重肝细胞内胆汁酸积聚，并加重胆汁淤积肝损害。.3.明确OATP3A1蛋白为肝细胞膜上胆酸排泌蛋白。. 通过构建经典的胆酸跨膜转运（Transwell）模型等实验多方面证实OATP3A1是一个新鉴定的、分布于肝细胞基底侧膜的关键胆酸排泌蛋白。.4.明确调控OATP3A1适应性高表达的分子调控机制。. 经生物信息学分析后发现：OATP3A1启动子区有丰富的顺式作用元件，构建相应的缺失突变启动子报告基因质粒，发现转录因子SP1和p65可分别结合在OATP3A1启动子-3478/-2578及-427/-183处顺式作用元件而上调其启动子活性；通过关键位点突变进一步证实上述位点发挥正调控作用；后经过一系列染色质免疫共沉淀实验再次确定SP1和p65可分别直接结合到OATP3A1启动子区-3453、-383处顺式作用元件上调其活性。.5.阐明OATP3A1适应性高表达具对抗胆汁淤积肝损害的分子机制。. 经过一系列转录调控实验，我们发现FGF19可以通过激活ERK和NFKB信号通路刺激SP1和p65表达而上调肝细胞基底侧膜OATP3A1的表达，增强肝细胞内胆汁酸外排；同时，FGF19也可通过ERK/SHP/CYP7A1信号轴抑制胆汁酸从头合成，最终减轻肝细胞内胆汁酸积聚达到对抗胆汁淤积的效应。上述研究加深了我们对胆汁淤积肝病发病分子机制的理解，也为其治疗提供新的关键靶点。