清胰汤对急性胆源性胰腺炎大鼠胰腺腺泡细胞Gpbar1信号传导通路的影响

Acute biliary pancreatitis (ABP) is a formidable disease, which, in severe forms, causes significant mortality. Pancreatic acinar cell injury induced by reflux of bile acids is the key and start step of ABP, but the mechanisms are still unknow. We reason that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G protein-coupled, cell surface, bile acid receptor. The presence of Gpbar1 on the apical pole of PACs was first identified by Perides et al. using C57BL/6 mice deleted for Gpbar1. They found that Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in taurolithocholic acid-induced pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to taurolithocholic acid So they concluded that Gpbar1 may play a critical role in the evolution of bile acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of ABP. The precise regulate mechanisms need to be further investigated. We devoted in study of the mechanism of acute biliary pancreatitis and therapeutic methods of integrated traditional and western medicine. We have established the ABP rat models in vivo and cell injury models in vitro by deoxycholic acid. Recently, we certified that the expression of Gpbar1 is correlation with the severity of disease, and Gpbar1 is expressed at pancreatic acinar cell membrane. Purpose of this project is to investigate: 1. The relationship between Gpbar1 expression and pancreatic acinar cell apoptosis/necrosis, and pancreatic enzymes activation. 2. By over-expression or silence-expression of Gpbar1 gene, to study its influence on the concentration of Ca2+ in acinar cell, and the activity of NF-κB. 3. To investigate the function and mechanism of Qingyi decoction on Gpbar1 signal transduction pathway from animal model, cell, and molecular levels. Through these studies, trying to clarify the role of bile acid-Gpbar1-cell molecules ([Ca2+]i, NF-κ B) signal transduction pathway in the pathogenesis of ABP and treatment mechanism of Qingyi decoction, to establish the new theoretical basis for further popularization use of Qingyi decoction in ABP.

逆行入胰管的胆汁酸诱导胰腺腺泡细胞损伤是急性胆源性胰腺炎（ABP）发病起始环节之一。Gpbar1是胆汁酸特异性细胞膜受体，其表达量同ABP严重程度密切相关。胆汁酸是否通过Gpbar1介导的信号传导通路诱导腺泡细胞损伤，诱发ABP？清胰汤可减轻胰腺腺泡细胞损伤而有效治疗ABP。清胰汤是否通过调控Gpbar1信号通路发挥治疗作用尚不明确。本课题拟通过ABP大鼠模型与体外细胞学实验研究：Gpbar1表达同胰腺腺泡细胞凋亡/坏死转换、胰酶激活的关系；建立Gpbar1过表达与沉默表达胰腺腺泡细胞系，检测双向调节Gpbar1对细胞钙离子浓度与NF-κB活性的影响；旨在从整体、组织、细胞和基因水平，应用共聚焦显微镜、流式细胞术、基因转染、凋亡检测、免疫细胞化学等方法，研究清胰汤对胰腺腺泡细胞Gpbar1信号通路调控作用，进一步探寻清胰汤治疗ABP的机制与作用靶点。为清胰汤进一步推广应用提供新的理论基础。

清胰汤是经多年临床实践和动物实验证实治疗急性胆源性胰腺炎的有效方剂，但详细机制尚不完全清楚。最近研究证实，胰腺腺泡细胞损伤是急性胆源性胰腺炎发病的起始环节与关键因素之一，急性胆源性胰腺炎可能是一种“受体介导疾病”。本研究应用激光扫描共聚焦显微镜、流式细胞术、基因转染、TUNEL、免疫细胞化学等方法，从动物整体、组织、细胞和基因水平观察急性胆源性胰腺炎时胆汁酸受体Gpbar1信号通路变化和清胰汤干预作用。.研究结果显示：①急性胆源性胰腺炎大鼠胰腺组织出现病理损伤，伴随Gpbar1基因表达升高，清胰汤干预组大鼠胰腺组织病理损伤明显减轻，Gpbar1基因表达出现下调。②在提取的大鼠原代胰腺腺泡细胞实验中，脱氧胆酸诱导的细胞损伤具有时效和量效关系，低浓度时主要表现为细胞凋亡，高浓度时出现细胞坏死，伴随Gpbar1蛋白表达改变，并同凋亡相关基因Bax、Bcl-2的表达变化相关。③应用流式细胞术检测不同浓度脱氧胆酸诱导胰腺腺泡细胞系AR42J细胞凋亡/坏死率实验，随着脱氧胆酸浓度递增（0.1mM—1.0mM）细胞凋亡/坏死率随之升高，具有明显的量效关系。④成功构建了Gpbar1基因过表达和Gpbar1基因RNAi慢病毒载体。⑤建立了Gpbar1过表达及干扰慢病毒感染细胞株。⑥Gpbar1过表达细胞系，脱氧胆酸诱导的胰腺腺泡细胞凋亡明显增多，伴随细胞内钙离子浓度和胞核NF-κB活性增高；Gpbar1沉默细胞系，脱氧胆酸诱导的胰腺腺泡细胞凋亡明显减少，伴随细胞内钙离子浓度和胞核NF-κB活性减低。⑦清胰汤可通过调控Gpbar1信号传导通路表达，减轻脱氧胆酸诱导的胰腺腺泡细胞损伤。.本研究结果提示：胆汁酸诱导的胰腺腺泡细胞损伤可能是急性胆源性胰腺炎发病机制中的重要环节，位于细胞膜表面的特异性受体Gpbar1 信号传导通路变化（钙离子、NF-κB）参与腺泡细胞损伤机制的调控，清胰汤可能通过调节Gpbar1信号传导通路减轻急性胆源性胰腺炎时胰腺腺泡细胞损伤，发挥治疗作用。为清胰汤的进一步推广应用奠定理论基础。