香青兰有效部位对OGD/R损伤诱导的人脑微血管内皮细胞程序性坏死的作用机制研究  

Effect of effective parts of Dracocephalum moldavica on the necroptosis of HBMECs after OGD/R injury

作　　者：杨志航 王雪萌 徐磊 苏文灵 卡德尔业·卡德尔 徐明[4] 郑瑞芳 邢建国 YANG Zhi-hang;WANG Xue-meng;XU Lei;SU Wen-ling;KADDER Kadeerye;XU Ming;ZHENG Rui-fang;XING Jian-guo(College of Pharmacy,Shihezi University,Shihezi 832000,China;Xinjiang Institute of Materia Medica,Urumqi 830002,China;Xinjiang Uyghur Medicine Key Laboratory,Urumqi 830002,China;China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]石河子大学药学院,新疆石河子832000 [2]新疆药物研究所,新疆乌鲁木齐830002 [3]新疆维吾尔药重点实验室,新疆乌鲁木齐830002 [4]中国药科大学,江苏南京210009

出　　处：《药学学报》2022年第2期409-418,共10页Acta Pharmaceutica Sinica

基　　金：新疆维吾尔自治区重点实验室开放课题(2020D04021);新疆维吾尔自治区自然科学基金项目(2020D01B50);2019年自治区高层次人才引进工程(柔性引进人才)项目;天山创新团队计划(2020D14011)。

摘　　要：本研究旨在探讨香青兰有效部位(effective parts of Dracocephalum moldavica,EPDM)通过抑制程序性坏死通路对缺血再灌注损伤人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMECs)的保护作用和分子机制。泛半胱氨酸天冬氨酸蛋白酶抑制剂Z-VAD-FMK联合氧糖剥夺/复氧(oxygen-glucose deprivation/re-oxygenation,OGD/R)损伤建立HBMECs程序性坏死模型,以模拟脑缺血再灌注损伤过程。细胞增殖及细胞毒性检测试剂盒(cell counting kit-8,CCK-8)检测细胞活力;Hoechst 33342/PI荧光双染检测细胞程序性坏死率;试剂盒法检测乳酸脱氢酶(lactate dehydrogenase,LDH)含量;采用2,7-二氯二氢荧光素二乙酸酯探针、钙黄绿素乙酰甲酯和JC-1探针分别检测细胞内活性氧(reactive oxygen species,ROS)、线粒体膜通透性转化孔(mitochondrial permeability transition pore,MPTP)开放情况以及线粒体膜电位(mitochondrial membrane potential,MMP);酶联免疫吸附实验检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和白细胞介素-6(interleukin-6,IL-6)释放情况;免疫印迹法检测程序性坏死相关蛋白表达。结果表明,与对照组比较,Z-VAD-FMK联合OGD/R可使HBMECs活力下降,程序性坏死率升高,LDH释放增加,ROS产生增多,MPTP开放,MMP降低,TNF-α、IL-1β以及IL-6分泌增加,受体相互作用蛋白3(receptor interacting protein kinase 3,RIP3)和线粒体丝氨酸/苏氨酸磷酸酶5(phosphoglycerate mutase 5,PGAM5)表达升高,磷酸化混合系结构域样蛋白(phospho-mixed lineage kinase domain-like protein,p-MLKL)/MLKL比值升高;而EPDM预保护可部分逆转这些因素的变化。上述结果表明,EPDM可能通过抑制RIP3/MLKL/PGAM5通路和MPTP开放以保护线粒体功能,进而使HBMECs免受脑缺血再灌注损伤,可为EPDM治疗脑缺血性相关疾病提供有价值的科学依据。We investigated the ability of Dracocephalum moldavica(EPDM)flavonoids to protect human brain microvascular endothelial cells(HBMECs)from necroptosis induced by ischemia-reperfusion injury.To mimic the process of cerebral ischemia-reperfusion injury,a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease(caspase)inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation(OGD/R)injury using HBMECs.Cell proliferation and cytotoxicity(cell counting kit-8,CCK-8)was used to measure cell viability.A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis.A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate.DCFH-DA probes,calcein AM and JC-1 probes were used to measure changes in ROS production,mitochondrial membrane permeability transformation pore(MPTP)opening and mitochondrial membrane potential(MMP),respectively.Enzyme-linked immunosorbent assay(ELISA)kits were chosen to detect the release of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).Western blotting was used to detect necroptosis-related proteins.The results show that relative to control group,Z-VAD-FMK combined with OGD/R injury reduced cell viability,increased the necroptosis rate and the levels of LDH and ROS in HBMECs.The MPTP of the model group cells opened and the MMP reduced.TNF-α,IL-1β,and IL-6 levels were significantly elevated.Furthermore,the expression of receptor-interacting protein kinase 3(RIP3)and mitochondrial phosphoglycerate mutase 5(PGAM5)was significantly increased,accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein(p-MLKL)/MLKL.EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury.These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function,thereby

关 键 词：香青兰有效部位 脑缺血再灌注损伤 氧糖剥夺/复氧 程序性坏死 混合谱系激酶结构域样蛋白 线粒体膜通透性转换孔 

分 类 号：R966[医药卫生—药理学]