胆汁酸通过激活NF-κB途径及破坏高尔基体结构上调肝纤维化/肝硬化患者血清GP73水平的机制研究

Golgi protein 73 (GP73) is a transmembrane glycoprotein that cleaved and released into the blood by the pre-metalloproteinase converting enzymes FURIN and others. Our and other laboratory studies have shown that elevated serum GP73 has a good diagnostic value of liver fibrosis/cirrhosis, but the mechanism is unknown. Our previous research revealed that the levels of serum GP73 and bile acids are positively correlated in patients with liver fibrosis/cirrhosis, and non-conjugated bile acid deoxycholate can transcriptionally upregulate the expression of endogenous GP73 and FURIN in hepatocytes. It has been reported that bile acids can activate the NF-κB pathway in hepatocytes, and there is a potential binding site of NF-κB in both the GP73 and FURIN gene promoter regions; The intracellular concentration of calcium ion affects the cleavage activity of FURIN; Bile acid can promote the secretion of GP73 in esophageal cancer cells by disrupting the Golgi apparatus. Based on these, it is speculated that bile acids may upregulate serum GP73 levels in patients with liver fibrosis/cirrhosis through these functions. Here we will use bile acids to stimulate different liver cancer cell lines and hepatic stellate cell line and construct a liver fibrosis mouse model fed by bile acids to clarify the molecular mechanisms of bile acids upregulating serum GP73 level through molecular and cellular experiments, which may provide a basis for serum GP73 in diagnosis of liver fibrosis/cirrhosis.

高尔基体蛋白73（GP73）是一种跨膜糖蛋白，可被前蛋白转化酶FURIN等切割释放入血。我们及其他的研究表明，血清GP73在肝纤维化/肝硬化患者中异常升高，但机制不详。前期结果发现，肝纤维化/肝硬化患者血清GP73与胆汁酸水平呈正相关；脱氧胆酸可上调肝细胞内源GP73和FURIN基因的表达。文献报道胆汁酸可激活NF-κB途径，且GP73和FURIN启动子区均有潜在NF-κB结合位点；FURIN的切割活性与胞内钙离子浓度有关；胆汁酸可破坏高尔基体结构促进食管癌细胞GP73分泌。据此推测，胆汁酸可能通过激活NF-κB通路，影响高尔基体结构和FURIN切割活性上调肝纤维化/肝硬化患者血清GP73水平。本课题拟利用胆汁酸刺激不同肝癌和肝星状细胞系，构建肝纤维化小鼠模型并饲喂胆汁酸，通过细胞和分子生物学实验，阐明胆汁酸上调GP73水平的分子机制，为血清GP73用于肝纤维化/肝硬化的辅助诊断提供依据。

慢性肝脏疾病严重危害人类生命健康。肝纤维化/肝硬化是不同病因慢性肝脏疾病的重要进展阶段，可被及时有效的临床干预所逆转。因此，研发可靠的诊断检测指标对于慢性肝病的管理至关重要。高尔基体蛋白73（Golgiprotein73，GP73）是细胞高尔基体上的一种跨膜蛋白，可被弗林蛋白酶（FURIN）等切割释放入血。多项研究表明，血清GP73的升高与肝脏疾病进展密切相关，有望作为慢性肝脏疾病的新型血清学标志物，但其相关机制不详。前期研究结果发现，肝纤维化/肝硬化患者血清GP73与胆汁酸水平呈正相关关系。. 本研究在细胞水平和动物水平探究了胆汁酸对GP73的影响，结果发现肝纤维化动物模型中血清GP73与血清胆汁酸同步升高；在Huh-7和SMMC7721细胞中，非结合型胆汁酸脱氧胆酸（DCA）处理后细胞上清GP73含量增加，而结合型胆汁酸牛磺胆酸（TCA）处理不影响上清GP73含量。DCA以一定的时间和剂量依赖性上调GP73的表达和释放；机制上，NF-κBP65可与GP73启动子直接结合，过表达NF-κBp65可致GP73启动子活性、mRNA水平和上清GP73含量均升高，抑制NF-κB通路后DCA对GP73释放的促进作用减弱。同样，在GP73启动子序列中突变NF-κB的结合位点，也会导致DCA对GP73启动子活性的促进作用下降。DCA灌胃小鼠实验也在体内水平证实了DCA通过NF-κB途径上调GP73表达；DCA处理和p65过表达可以在Huh7和SMMC7721细胞上调GP73切割酶Furin的启动子活性和mRNA水平，但仅在SMMC7721细胞中能观察到蛋白水平升高。Furin及其家族成员PCSK6、PCSK8对GP73无切割作用，也不影响其分泌；除了上调GP73的表达促进其释放外，DCA还可以引起高尔基体结构的破坏从而促进GP73释放。. 综上，肝病状态下血清GP73水平的异常升高与患者血清胆汁酸浓度增加有关，DCA可以通过激活NF-κB通路及破坏高尔基体结构上调GP73的表达和释放。阐明了胆汁酸调控GP73的分子机制，为血清GP73用于肝纤维化/肝硬化的临床辅助诊断提供了依据。

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