目的 研究田蓟苷抗脑缺血再灌注损伤程序性坏死的作用及机制。方法 人神经母细胞瘤细胞SH-SY5Y用Caspase抑制剂Z-VAD-FMK预处理16 h后缺氧培养6 h、复氧培养2 h建立脑缺血再灌注程序性坏死模型，在缺氧前6 h给予田蓟苷进行干预。采用Hoechst33342/PI染色检测坏死细胞数目；采用CCK-8法检测细胞活力；采用ELISA法检测细胞上清中乳酸脱氢酶（lactate dehydrogenase，LDH）活性和白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平及细胞内活性氧（reactive oxygen species，ROS）水平和线粒体通透性转换孔（mitochondrial permeability transition pore，mPTP）的变化；采用荧光显微镜检测细胞线粒体膜电位变化；采用Western blotting法检测细胞程序性坏死通路相关蛋白如受体相互作用蛋白3（receptor interacting protein kinase 3，RIPK3）、混合系结构域样蛋白（mixed lineage kinase domain-like protein，MLKL）、钙/钙调蛋白依赖性激酶II（calcium/calmodulin-dependent protein kinase Ⅱ，CaMKⅡ）、磷酸甘油酸变位酶5（phosphoglycerate mutase family 5，PGAM5）表达情况。结果 与模型组比较，田蓟苷组细胞存活率显著提高（P<0.05）；细胞上清中LDH活性和IL-1β、IL-6、TNF-α水平均显著降低（P<0.05）；细胞内ROS水平显著降低（P<0.05）；细胞线粒体膜电位显著升高（P<0.05），mPTP开放被显著抑制（P<0.05）；细胞RIPK3、MLKL、CaMKⅡ和PGAM5蛋白表达水平均显著降低（P<0.05）。结论 田蓟苷可能通过抑制程序性坏死途径，保护线粒体，从而发挥抑制脑缺血再灌注损伤的作用。

Objective To study the effect and mechanism of tilianin against programmed necrosis of cerebral ischemia-reperfusion injury. Methods Human neuroblastoma cells SH-SY5Y were pretreated with Caspase inhibitor Z-VAD-FMK for 16 h, then cultured under hypoxia for 6 h and reoxygenated for 2 h to establish a model of programmed cerebral ischemia-reperfusion necrosis. Tilianin was given for intervention 6 h before hypoxia. Hoechst33342/PI staining was used to detect the number of necrotic cells; CCK-8 method was used to detect cell viability; ELISA method was used to detect lactate dehydrogenase (LDH) activity and interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) levels in supernatant, intracellular reactive oxygen species (ROS) level and mitochondrial permeability transition pore (mPTP) changes in SH-SY5Y cells; Fluorescence microscopy was used to detect changes in cell mitochondrial membrane potential; Western blotting was used to detect expressions of receptor interacting protein kinase 3 (RIPK3), mixed-line domain-like proteins (MLKL), calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and phosphoglycerate mutase family 5 (PGAM5). Results Compared with model group, cell viability of tilianin group was significantly increased (P < 0.05); LDH activity and levels of IL-1β, IL-6, TNF-α in supernatant were significantly decreased (P < 0.05); ROS level were significantly reduced (P < 0.05); Cell mitochondrial membrane potential was significantly increased (P < 0.05); Mitochondrial membrane permeability transition pore (mPTP) opening was significantly inhibited (P < 0.05); Expressions of RIPK3, MLKL, CaMKⅡ and PGAM5 were significantly decreased (P < 0.05). Conclusion Tilianin may protect mitochondria by inhibiting programmed necrosis pathway, thereby inhibiting cerebral ischemia-reperfusion injury.

R285.5

新疆维吾尔自治区自然科学基金（青年基金）资助项目（2020D01B50）；新疆维吾尔自治区公益性科研院所基本科研业务经费资助项目（KY2020086）；2019年新疆维吾尔自治区高层次人才引进工程（柔性引进人才）项目