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Abstract：
The mitochondrial calcium uniporter (MCU) is a major protein for the uptake of mitochondrial calcium to regulate intracellular energy metabolism, including processes such as mitophagy. The present study investigated the effect of the MCU on mitophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP) in vitro. The normal human PDECs (HPDE6-C7) were treated with caerulein (CAE) to induce AP-like changes, with or without ruthenium red to inhibit the MCU. The mitochondrial membrane potentials (MMPs) and mitochondrial Ca2+ levels were analyzed by fluorescence. The expression levels of MCU, LC3, p62, and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), putative kinase 1 (PINK1), and Parkin were measured by western blotting and immunofluorescence. Mitophagy was observed by confocal fluorescence microscopy and transmission electron microscopy. The results showed that CAE increased the MCU protein expression, mitochondrial Ca2+ levels, MMP depolarization and the protein expression of mitophagy markers including the LC3II/I ratio, PINK1, and Parkin. CAE decreased the protein expression of p62 and TOMM20, and promoted the formation of mitophagosomes in HPDE6-C7 cells. Notably, changes in these markers were reversed by inhibiting the MCU. In conclusion, an activated MCU may promote mitophagy by regulating the PINK1/Parkin pathway in PDECs in AP.
摘要：
线粒体钙离子转运蛋白(MCU)是摄取线粒体钙调控细胞内能量代谢的主要蛋白,包括线粒体自噬等过程。本研究研究了MCU对急性胰腺炎（AP）胰腺导管上皮细胞（PDECs）线粒体自噬的影响。正常人PDECs（HPDE6-C7）用caerulein（CAE）处理以诱导AP样变化，有或没有钌红抑制MCU。通过荧光分析线粒体膜电位（MMPs）和线粒体Ca2水平。MCU的表达水平，LC3，p62和外线粒体膜复合物亚基20（TOMM20）的转位酶，推定激酶1（PINK1），和Parkin通过蛋白质印迹和免疫荧光进行测量。通过共聚焦荧光显微镜和透射电子显微镜观察线粒体自噬。结果表明，CAE增加了MCU蛋白的表达，线粒体Ca2+水平，MMP去极化和线粒体自噬标志物包括LC3II/I比值的蛋白表达,PINK1和Parkin.CAE降低了HPDE6-C7细胞中p62和TOMM20的蛋白表达，并促进了有丝分裂体的形成。值得注意的是,这些标志物的变化通过抑制MCU而逆转。总之,激活的MCU可能通过调节APPDEC中的PINK1/Parkin通路来促进线粒体自噬。
