胰腺腺泡细胞线粒体通透性转换诱导自噬在急性胰腺炎中的作用及机制

Acute pancreatitis is an inflammatory disorder of the exocrine pancreas, but its pathogenesis remains poorly understood. Autodigestion of the pancreas by its own premature activation of digestive proteases induced by autophagic dysfunctions is thought to be an critical event in the onset of acute pancreatitis. Mitochondrial permeability transition (MPT) results in mitochondrial depolarization and loss of ATP production, which has been found to induce autophagy in several cells, eg. cardiomyocytes and hepatocytes and is of vital importance for the fate of cells. Gukovsky I, et al. in their review directed the roles for mitochondrial and autophagic dysfunctions in the pathogenesis of pancreatitis. But, none of published research demostrating MPT-induced autophagy in the pathogenesis of acute pancreatitis and its exact molecular mechanism can be currently available, indicating an urgent necessity for detailed investigations on this issue. In the previous study, we found MPT critical in pathological response to cholecystokinin-8 (CCK-8) in experimental acute pancreatitis. MPT lead to loss of mitochondrial membrane potential, metabolism and ATP production, digestive enzyme activation and necrosis. When CCK-8-stimulated acinar MPT was blocked in pancreatic acinar cells by pharmacological inhibitor cyclosporin A (CsA), which binds to and inhibits cyclophilin D, mitochondrial function was sustained, digestive enzyme activation minimized, necrosis prevented and inflammatory responses diminished. Therefore, we hypothesize that the mechanism underlying this linkage implicates MPT-activated autophagy and impaired autophagy in trypsinogen activation and necrosis, which is crucial for triggering acute pancreatitis. To verify this hypothesis, firstly we plan to test the effects of CsA-inhibited MPT on autophagy induction, trypsinogen activation and cell necrosis in CCK-8 stimulated changes in pancreatic acinar AR42J cells, in which autophagy enhancer rapamycin will be also used to confirm autophagy activation. To explore the mechanism of MPT-induced autophagy in acute pancreatitis, the molecular changes of AMPK/mTOR signal pathway, in which the AMP-activated protein kinase (AMPK) senses energy deprivation and induce autophagy via mTOR inhibition, will be detected in AR42J cells stimulated by CCK-8 and administrated by CsA. To ascertain "energy sensor" AMPK responses to MPT in triggering autophagy pathway, the effect of AMPK inhibitor Compound C on autophagy will be determined. In conclusion, the results of the study will elucidate the innovative linkage and mechanism of MPT and autophagy in the pathogenesis of acute pancreatitis. Thus, the project is of great value in exploring, on the new aspect, upper-stream mechanism of autophagy induction and the pathogenesis of acute pancreatitis, and providing new method and target for the therapy of this disease.

胰腺腺泡细胞异常自噬激活酶原是公认的急性胰腺炎(AP)发病的始动环节，但诱导自噬的上游机制尚未阐明。我们前期研究发现，腺泡细胞发生线粒体通透性转换(MPT)，线粒体去极化和ATP下降，可导致细胞内胰酶活性升高和细胞坏死；MPT抑制剂环孢素A(CsA)能改善线粒体功能，降低胰酶活性，减轻细胞坏死。因此，我们提出假说"MPT诱导自噬是促进AP发生发展的重要环节"。为证明此假说，本课题将用胆囊收缩素(CCK-8)刺激大鼠胰腺腺泡细胞株AR42J造成AP细胞模型，观察CsA抑制MPT对自噬、酶原活化以及细胞坏死的影响；由于AMPK/mTOR途径与ATP降低诱导自噬的关系最为密切，因此本课题以此为切入点，进一步研究该途径中关键信号分子的变化，以探讨MPT诱导自噬的机制。上述研究内容均未见论文报道。本课题可望阐述自噬诱导的新机制和AP发病机制的新理论，为AP的有效防治提供新思路和药物作用新靶点。

研究腺泡细胞内线粒体异常以及自噬异常有助于阐明急性胰腺炎（AP）发病的始动机制。本研究通过雨蛙肽（Caerulein， CER）小鼠AP模型和CCK-8（雨蛙肽类似物）刺激AR42J细胞实验，从AMPKα-CyPD-mTOR途径探索了AP过程中发生自噬的机制：1）AP时AMPKα、CyPD、mTOR表达水平增强，自噬蛋白Beclin1、Atg12、LC3-II、ULK1的表达也增强，说明胰腺炎症可激活AMPKα—CyPD—mTOR信号通路，诱导自噬活化；2）线粒体通透性转换孔抑制剂CsA能够抑制CyPD的表达，并抑制AMPKα/mTOR信号和自噬的过程；3）CCK-8表达刺激AR42J细胞，自噬流增强，自噬增强剂Rapamycin可以增强自噬水平，而CsA在细胞水平抑制自噬的作用不明显，待进一步研究。4) 观察电镜的“金标准”电镜结果显示，AP小鼠胰腺腺泡细胞固缩，线粒体形态失常，自噬体内含未降解的线粒体、酶原颗粒、内质网、碎片等物质，CsA处理后上述自噬表现程度减轻。5）通过microRNA芯片筛选了AP自噬相关的miR-216a等特定microRNA，进一步研究发现miR-216a能够反映胰腺损伤和AP病情严重程度，与自噬活性趋势呈负相关。本课题阐述了自噬诱导的新机制和AP发病机制的新理论，为AP的有效防治提供新思路和药物作用新靶点。