田蓟苷抗脑缺血再灌注程序性坏死的作用及机制研究

Effect and mechanism of tilianin against necroptosis on cerebral ischemia-reperfusion

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,m PTP)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 kinaseⅡ,Ca MKⅡ)、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),m PTP开放被显著抑制(P<0.05);细胞RIPK3、MLKL、Ca MKⅡ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-SY5 Y 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-SY5 Y 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, Ca MKⅡ and PGAM5 were significantly decreased(P < 0.05). Conclusion Tilianin may protect mitochondria by inhibiting programmed necrosis pathway, thereby inhibiting cerebral ischemia-reperfusion injury.