目的 采用泛半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid protease,Caspase)抑制剂Z-VAD-FMK联合氧糖剥夺/复氧(oxygen-glucose deprivation/re-oxygenation, OGD/R)建立体外脑缺血再灌注损伤模型,探讨了香青兰有效部位(effective parts of Dracocephalum moldavica, EPDM)对人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMECs)的保护作用。方法 CCK-8法检测细胞活力,倒置显微镜下观察细胞形态,NO测定试剂盒检测NO含量,transwell法检测细胞迁移情况,免疫荧光检测血管内皮-钙黏蛋白(VE-Cadherin)的表达情况,免疫印迹检测酪氨酸激酶(SRC)、内皮素-1(ET-1)、紧密连接蛋白Claudin 5、内皮性一氧化氮合酶(eNOS)、及磷酸化eNOS的蛋白表达。结果 与对照组比较,Z-VAD-FMK联合OGD/R可使HBMECs活力下降,细胞形态变差,细胞迁移减少,NO释放量降低,VE-cadherin表达减少,Src、Claudin 5、磷酸化eNOS表达减少,ET-1表达升高;而EPDM预处理可部分逆转Z-VAD-FMK联合OGD/R引起的上述因素的变化。结论 EPDM对体外脑缺血再灌注模型下的HBMECs具有保护作用,可促进细胞增殖、迁移,增强血管舒缩调节能力,并通过增强紧密连接和黏附连接维持屏障功能。
Abstract
OBJECTIVE To investigate the protective effect of EPDM on endothelial cells, a cerebral ischemia-reperfusion injury model established by treatment with the pan-cysteine aspartic acid protease(Caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation(OGD/R). METHODS Cell Counting Kit-8(CCK-8) was used to detect cell viability, cell morphology was observed by microscope, NO content was detected by NO determination kit, and cell migration was detected by Transwell method. The expression of vascular endothelium-cadherin was detected by immunofluorescence. Western blot was used to detect the protein expressions of non-receptor tyrosine kinase(Src), endothelin-1(ET-1), blood-brain barrier tight junction associated protein 5(Claudin 5), endothelial nitric oxide synthase(eNOS), and phosphorylated eNOS. RESULTS Compared with the control group, Z-VAD-FMK combined with OGD/R reduced HBMECs activity, cell morphology, cell migration, NO release, VE-cadherin expression, SRC, Claudin 5 and p-eNOS expression, andincrease ET-1 expression. EPDM pretreatment can partially reverse the above factors induced by Z-VAD-FMK combined with OGD/R. CONCLUSION EPDM has a protective effect on HBMECs under cerebral ischemia reperfusion model, which can promote cell proliferation and migration, enhance vasomotor regulation, and maintain barrier function by enhancing tight junctions and adhesion junctions.
关键词
香青兰有效部位 /
人脑微血管内皮细胞 /
脑缺血再灌注损伤 /
氧糖剥夺/复氧 /
屏障功能
{{custom_keyword}} /
Key words
effective parts of Dracocephalum moldavica /
human brain microvascular endothelial cell /
ischemia-reperfusion injury /
oxygen-glucose deprivation/re-oxygenation /
barrier function
{{custom_keyword}} /
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] WANG K, LIU F, LIU C Y, et al. The long noncoding RNA NRF regulates programmed necrosis and myocardial injury during ischemia and reperfusion by targeting miR-873[J]. Cell Death Differ, 2016, 23(8): 1394-1405.
[2] ALMAGROM C D, TATIANA G, IZRAEL-TOMASEVIC A, et al. Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death[J]. Cell Death Differ, 2017, 24(1): 26-37.
[3] ZHU P J, HU S Y, JIN Q H, et al. Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: a mechanism involving calcium overload/XO/ROS/mPTP pathway[J]. Redox Biol, 2018, 16: 157-168.
[4] ZHOU H, ZHU P J, GUO J, et al. Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury[J]. Redox Biol, 2017, 13: 498-507.
[5] YU N, CHEN W, XING J G, et al. HPLC determination of four components in Yixin Badiranjibuya granules[J]. Chin J Pharm Anal(药物分析杂志), 2015, 35(4): 670-674.
[6] ZHENG R F, DU Y W, ZENG C, et al. Total flavones of Dracocephalummoldavica L. protect astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway[J]. BMC Complement Med Ther, 2020, 20(1): 78.Doi: 10.1186/s12906-020-2846-4.
[7] SUN Y, LIU T, DAI X, et al. Effect of total flavones of DracocephalummoldavicaL. on inflammation induced by transient cerebral ischemia[J]. Acta Biophys Sin(生物物理学报), 2012, 28(6): 469-476.
[8] MA L Y, ZENG C, ZHENG R F, et al. Protective effects of tilianin on brain tissue in cerebral ischemia-reperfusion injury model rats[J]. J China Pharm(中国药房), 2018, 29(20): 2805-2810.
[9] WU P, YAN X S, ZHOU L L, et al. Involvement of apoptosis in the protective effects of Dracocephalummoldavaica in cerebral ischemia reperfusion rat model[J]. J Toxicol Environ Health A, 2019, 82(19): 1036-1044.
[10] XING J, HE C, YU N, et al. The preparation method and application of the Dracocephalummoldavica extracts: China, CN201510652848.7[P]. 2016-01-20.
[11] ZHANG Y H, ZHANG X C, PARK T S, et al. Cerebral endothelial cell apoptosis after ischemia-reperfusion: role of PARP activation and AIF translocation[J]. J Cereb Blood Flow Metab, 2005, 25(7): 868-877.
[12] LAKHANS E, KIRCHGESSNER A, HOFER M. Inflammatory mechanisms in ischemic stroke: therapeutic approaches[J].J Transl Med, 2009,7:97.Doi: 10.1186/1479-5876-7-97.
[13] HALESTRAP A P. A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection[J].Biochem Soc Trans, 2010, 38(4):841-860
[14] YU Q J,Tao H,WANG X,et al.Targeting brain microvascular endothelial cells:a therapeutic approach to neuroprotection against stroke[J].Neural Regen Res,2015,10(11):1882-1891.
[15] LIU D, YU G Z, SONG J G. Astragaloside Ⅳ alleviates the effects of hypoxia on brain microvascular endothelial cells apoptosis and Inflammation by down-regulating miR-327[J]. Chin Pharm J(中国药学杂志),2021,56(19):1571-1576.
[16] ZHANG J J, LIU T, WANG Y Q, et al.The influence of transforming growth factor β1 on barrier function of endothelial cells[J].Med J Qilu(齐鲁医学杂志),2007,22(4):297-299+302.
[17] YANG A Y, WEN T, WU H A, et al.Comparative study of the effectsof different magnetic nanomaterials on vascular endothelial cells[J].Beijing Biomed Eng(北京生物医学工程),2018,37(3):235-240.
[18] ZHOU H, LI D D, ZHU P J, et al. Inhibitory effect of melatonin on necroptosis via repressing the Ripk3-PGAM5-CypD-mPTP pathway attenuates cardiac microvascular ischemia-reperfusion injury[J]. J Pineal Res, 2018, 65(3):e12503. Doi: 10.1111/jpi.12503.
[19] GUO Y Q, ZHAO H, CHEN J, et al.The role of Claudin-5 mediated BBB dysfunction in cerebral ischemia reperfusion[J].Chin J Cell Biol(中国细胞生物学学报),2021,43(7):1536-1542.
[20] YAKUBU M A, LEFFLER C W. Regulation of ET-1 biosynthesis in cerebral microvascular endothelial cells by vasoactive agents and PKC[J].Am J Physiol, 1999,276(2):C300-C305.
[21] CHEN Y, LIU D, LI L Z, et al. Effects of potentilla anserina L. Polyphenols on NO, NOS, ET-1 and HIF-1α in human umbilical vein endothelial cells injured by glucose and oxygen deprivation[J].China Pharm (中国药师),2019,22(3):389-394.
[22] NOGUCHI N, TERAMOTO K, OCHIAI T, et al. The close participation of ET-1 and NO in human hepatic hemodynamics[J]. Hepatol Res, 2006,36(2):86-93.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
新疆维吾尔自治区青年科技人才-乡村振兴项目资助(WJWV-XCzX202205);中央引导地方科技发展专项资助(ZYYD2022A02);新疆维吾尔自治区重点实验室开放课题资助(2020D04020);新疆维吾尔自治区公益性科研院所科研业务费资助(KY2018137);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)项目资助
{{custom_fund}}
PDF(1973 KB)
