高良姜素通过激活Nrf2信...氧_复氧诱导的心肌细胞损伤_杨芳.pdf

基金项目:陕西省重点研发计划项目(2021SF 396);陕西省中医药科研项目(2021 ZZ JC042);陕西省中医药管理局市级中医医院科研能力提升项目(SZY NLTL 2022 011);咸阳市重点研发计划项目(L2022ZDYFSF076)作者简介:杨芳，女，1986 08 生，硕士，主管药师，E-mail:yangf87126 com收稿日期:2023 01 13高良姜素通过激活 Nrf2 信号通路减轻低氧/复氧诱导的心肌细胞损伤杨芳1，刘宝贵1，庞洋1，贾帅芸1，廉秋芳2*(1延安大学咸阳医院药学部，咸阳712000;2延安大学咸阳医院心内科;*通讯作者，E-mail:wjylqf163 com)摘要:目的观察高良姜素(galangin，Gal)对低氧/复氧(hypoxia/reoxygenation，H/)诱导的心肌细胞损伤的影响，并探讨可能的作用机制。方法采用小鼠 HL-1 心肌细胞构建 H/细胞模型。实验分为常氧对照组、H/组(先低氧 6 h，后常氧12 h)、H/+溶剂组、H/+10 mol/L Gal 组、H/+30 mol/L Gal 组和 H/+50 mol/L Gal 组。H/+溶剂组、H/+10 mol/LGal 组、H/+30 mol/L Gal 组和 H/+50 mol/L Gal 组分别采用溶剂对照二甲基亚砜、10 mol/L Gal、30 mol/L Gal、50 mol/L Gal 预处理心肌细胞 1 h，然后进行 H/处理。采用细胞计数试剂盒 8(cell counting kit-8，CCK-8)检测细胞活性。采用乳酸脱氢酶(lactate dehydrogenase，LDH)试剂盒检测细胞损伤。采用 TUNEL 实验检测细胞凋亡。采用活性氧(reactiveoxygen species，OS)试剂盒检测 OS 水平。采用丙二醛(malondialdehyde，MDA)试剂盒检测 MDA 含量。采用超氧化物歧化酶(superoxide dismutase，SOD)试剂盒检测 SOD 活性。采用荧光素酶报告基因实验检测核转录因子 E2 相关因子 2(nuclearfactor-erythroid 2-related factor 2，Nrf2)的转录活性。采用 Western blot 检测 Nrf2、醌氧化还原酶 1(quinone oxidoreductase 1，NQO-1)和血红素加氧酶 1(heme oxygenase 1，HO-1)的蛋白表达水平。通过 GraphPad Prism 8 软件计算高良姜素的半最大效应浓度(half maximal effective concentration，EC50)和半最大抑制浓度(half maximal inhibitory concentration，IC50)。采用 Nrf2 抑制剂 ML385 验证高良姜素是否通过 Nrf2 抑制 H/心肌细胞损伤。结果与常氧对照组比较，H/组和 H/+溶剂组中心肌细胞活性降低，而心肌细胞损伤、凋亡、OS 水平和 MDA 含量升高，并且 SOD 活性降低，差异均有统计学意义(P 0 01)。与 H/组和 H/+溶剂组比较，不同剂量的高良姜素处理组心肌细胞活性均升高(P 0 05)，而心肌细胞损伤、凋亡和氧化应激均下降，并呈剂量依赖性，差异有统计学意义(P 0 05)。高良姜素增加 H/处理的心肌细胞活性的 EC50值为35 8 mol/L，减少 H/诱导的细胞损伤的 IC50值为 38 3 mol/L，减少 H/诱导的细胞凋亡的 IC50值为 25 8 mol/L，减少H/诱导的 OS 产生的 IC50值为26 7 mol/L，减少 H/诱导的 MDA 产生的 IC50值为23 6 mol/L，增加 H/处理的心肌细胞 SOD 活性的 EC50值为 25 4 mol/L。与 H/组和 H/+溶剂组比较，不同剂量的高良姜素处理组 Nrf2 的细胞核表达量和转录活性均增加，NQO-1 和 HO-1 表达水平均上升，并呈剂量依赖性，差异有统计学意义(P 0 05)。抑制 Nrf2 显著逆转高良姜素对 H/诱导的心肌细胞损伤的保护作用(P0 05)。结论高良姜素通过激活 Nrf2 信号通路减轻 H/诱导的心肌细胞损伤。关键词:心肌细胞;低氧/复氧;高良姜素;Nrf2;缺血再灌注损伤中图分类号:5422文献标志码:A文章编号:1007 6611(2023)04 0459 11DOI:1013753/j issn1007 6611202304008Galangin ameliorates hypoxia/reoxygenation-induced injury in cardiomyocytes by enhancing Nrf2 signaling pathwayYANG Fang1，LIU Baogui1，PANG Yang1，JIA Shuaiyun1，LIAN Qiufang2*(1Department of Pharmacy，Xianyang Hospital of Yan anUniversity，Xianyang 712000，China;2Department of Cardiology，Xianyang Hospital of Yan an University;*Corresponding author，E-mail:wjylqf163 com)Abstract:ObjectiveTo observe the effect of galangin(Gal)on hypoxia/reoxygenation(H/)-induced injury in cardiomyocytes andexplore the potential underlying mechanismMethodsThe mouse HL-1 cardiomyocytes were applied to establish the H/cell modelHL-1 cardiomyocytes were divided into normoxic control group，H/group(hypoxia for 6 h and then normoxia for 12 h)，H/+vehiclegroup，H/+10 mol/L Gal group，H/+30 mol/L Gal group，and H/+50 mol/L Gal group HL-1 cells in H/+vehiclegroup，H/+10 mol/L Gal group，H/+30 mol/L Gal group，and H/+50 mol/L Gal group were pretreated with dimethyl-sulfoxide，10 mol/L Gal，30 mol/L Gal，and 50 mol/L Gal for 1 h，and then treated with H/，respectively Cell viability wasdetected by cell counting kit-8(CCK-8)assay Cell injury was measured by lactate dehydrogenase(LDH)assay Cell apoptosis wasexamined by TUNEL assay eactive oxygen species(OS)level was assessed by OS assay Malondialdehyde(MDA)content wasevaluated by MDA assay Superoxide dismutase(SOD)was detected by SOD assay The transcriptional activity of nuclear factor-erythroid 2-related factor 2(Nrf2)was monitored by luciferase reporter assay Protein expression of Nrf2，quinone oxidoreductase 1954山西医科大学学报，2023 年 4 月，第 54 卷 第 4 期(NQO-1)and heme oxygenase-1(HO-1)was detected by Western blot Half maximal effective concentration(EC50)and half maximalinhibitory concentration(IC50)of Gal were calculated by GraphPad Prism 8 software Nrf2 inhibitor ML385 was utilized to confirmwhether Gal inhibites H/-induced cardiomyocyte injury via Nrf2esultsCompared with normoxic control group，the cell viabilitywas significantly decreased，the cell injury，the apoptosis，OS level and MDA content were significantly increased，and SOD activitywas significantly reduced in H/group and H/+vehicle group(all P0 01)Compared with H/group and H/+vehicle group，the cell viability was significantly increased in different Gal groups(P 0 05)，and the cell injury，the apoptosis and the oxidativestress were decreased in H/-exposed cardiomyocytes in a dose-dependent manner(P 0 05)The EC50of Gal in increasing theviability of H/-treated cardiomyocytes was 35 8 mol/L The IC50of Gal in decreasing H/-induced cardiomyocyte injury was 38 3mol/L The IC50of Gal in decreasing H/-induced OS production was 6 7 mol/L The IC50of Gal in decreasing H/-inducedMDA production was 23 6 mol/L The EC50of Gal in increasing the SOD activity in H/-treated cardiomyocytes was 25 4 mol/LCompared with H/group and H/+vehicle group，the level of Nrf2 in nucleus was significantly increased in different Gal groups，and the transcriptional activity of Nrf2 was enhanced，and the expression of NOQ-1 and HO-1 was increased in a dose-dependent man-ner(P0 01)Inhibition of Nrf2 reversed the pr