【摘要】 基于PTEN诱导激酶1(PTEN-induced kinase 1, PINK1)/Parkin信号通路探讨黄杞苷(engeletin, Eng)对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation, OGD/R)诱导的神经元自噬的影响。以小鼠海马神经元细胞系HT22为研究对象，建立OGD/R诱导的HT22细胞损伤模型。将HT22细胞分为模型组(OGD/R组), Eng低、中、高剂量组(Eng-L、-M、-H组，分别使用1、 5、 10 mmol/L剂量), si-PINK1 NC+Eng-H组，si-PINK1+Eng-H组和空白对照组(control组)。对细胞活性，细胞凋亡，活性氧类(reactive oxygen species, ROS)含量，谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px),丙二醛(malondialdehyde, MDA)和超氧化物歧化酶(superoxide dismutase, SOD)水平，线粒体膜电位变化，细胞自噬水平，PINK1、 E3泛素连接酶Parkin,微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3, LC3),泛素结合蛋白p62及caspase3的表达等指标加以评估。结果显示，与control组比较，OGD/R组的HT22细胞活性，GSH-Px、 SOD水平，线粒体膜电位，细胞自噬水平，蛋白LC3-Ⅱ/LC3-Ⅰ比值，PINK1和Parkin蛋白表达均显著下降，而细胞凋亡率，caspase3蛋白表达水平，ROS、 MDA含量和p62蛋白表达量均显著增加(均P<0.05);与OGD/R组比较，Eng-L、-M、-H组HT22细胞活性，GSH-Px、 SOD活性，线粒体膜电位，自噬水平，蛋白LC3-Ⅱ/LC3-Ⅰ比值、 PINK1和Parkin蛋白表达均显著增加，而细胞凋亡率，caspase3蛋白表达，ROS、 MDA含量和p62蛋白表达量均显著下降(均P<0.05);与Eng-H组比较，si-PINK1+Eng-H组可以逆转Eng-H对HT22细胞的自噬增强作用，加剧细胞损伤和凋亡。由此，研究首次发现Eng可以上调PINK1/Parkin信号通路表达，通过线粒体自噬途径减轻OGD/R诱导的神经元损伤。更多还原

【Abstract】 The aim of this study was to investigate the effect of engeletin(Eng) on the autophagy of neurons induced by oxygen-glucose deprivation/reoxygenation(OGD/R) by the PTEN-induced kinase 1(PINK1)/Parkin signaling pathway. The mouse hippocampal neuron HT22 cell line was used as the study object to establish an OGD/R-induced cell damaged model. HT22 cells were grouped into the model group(OGD/R group), the low-, medium-and high-dose Eng groups(Eng-L,-M,-H groups, with 1, 5, 10 mmol/L Eng, respectively), the si-PINK1 NC+Eng-H group, the si-PINK1+Eng-H group, and blank group(control group). The cell viability, cell apoptosis, reactive oxygen species(ROS) level, the cell levels of glutathione peroxidase(GSH-Px), malondialdehyde(MDA), and superoxide dismutase(SOD),the changes of mitochondrial membrane potential, cell autophagy level, the expressions of PINK1, Parkin(E3 ubiquitin ligase), microtubule-associated protein 1 light chain 3(LC3), ubiquitin-binding protein p62, and caspase3 were evaluated using appropriate methods. The results showed that compared to those of the control group, the viability of HT22 cells, the GSH-Px, the SOD levels, the mitochondrial membrane potential, the cell autophagy level, the ratio of LC3-Ⅱ/LC3-Ⅰ, and the protein expressions of PINK1 and Parkin in the OGD/R group were significantly decreased(all with P<0.05), whereas the apoptosis rate, the caspase3 protein expression, ROS, MDA levels, and p62 protein expression all significantly increased(all with P<0.05). Compared to those of the OGD/R group, the viability of HT22 cells, the GSH-Px, SOD levels, the mitochondrial membrane potential, the cell autophagy level, the ratio of LC3-Ⅱ/LC3-Ⅰ, and the protein expressions of PINK1 and Parkin in the Eng-L、-M and-H groups were significantly increased(all with P<0.05) while the apoptosis rate, the caspase3 protein expression, ROS, MDA levels and the p62 protein expression all significantly decreased(all with P<0.05). Compared to the Eng-H group, si-PINK1+Eng-H treatment reversed the effect of Eng-H on the enhancement of autophagy of HT22 cells, and aggravated cell injury and apoptosis. In conclusion, we have firstly demonstrated that Eng can up-regulate the expression of PINK1/Parkin signaling pathway, and alleviate OGD/R-induced neuronal damage through the mitochondrial autophagy pathway.更多还原