柿叶黄酮提取物对氧糖剥夺/复糖复氧损伤的HT22细胞氧化应激的保护作用

doi:10.6040/j.issn.1671-7554.0.2017.1234

摘要/Abstract

摘要： 目的探讨柿叶黄酮提取物(FLDK)对氧糖剥夺/复糖复氧(OGD/R)损伤的HT22细胞氧化应激的影响及其可能机制。方法将HT22细胞随机分为正常对照组(Control组)、OGD/R组、OGD/R+FLDK治疗组(OGD/R+FLDK组)、OGD/R+FLDK+转录因子NF-E2相关因子(Nrf2)基因沉默组(OGD/R+FLDK+si-Nrf2组)。对HT22细胞氧糖剥夺3 h并复糖复氧24 h建立OGD/R模型;FLDK为自氧糖剥夺开始即加入,一直持续到复糖复氧结束;利用si-Nrf2阻断Nrf2通路。采用CCK-8法检测细胞活性,乳酸脱氢酶(LDH)法测定细胞损伤率,试剂盒法测定丙二醛(MDA)含量及超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,2',7'-二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测胞内活性氧(ROS)水平,Western blotting法检测Nrf2及血红素氧合酶-1(HO-1)蛋白表达。结果与Control组相比,OGD/R组细胞活性下降(P<0.001),细胞损伤率升高(P<0.001),氧化产物ROS及MDA含量增加(P<0.001),抗氧化酶SOD及GSH-Px活性下降(P<0.001),全细胞HO-1蛋白表达升高(P=0.009)及胞核Nrf2蛋白表达升高(P=0.016);与OGD/R组相比,FLDK治疗可使细胞活性升高(P<0.001),细胞损伤率降低(P<0.001),氧化产物ROS及MDA含量下降(P<0.001),抗氧化酶SOD及GSH-Px活性升高(P<0.001),全细胞HO-1蛋白表达升高(P<0.001)及核内Nrf2蛋白表达升高(P=0.005);与OGD/R+FLDK组相比,进行Nrf2基因沉默预处理后,HO-1蛋白表达下降(P<0.001)及胞核Nrf2蛋白表达下降(P<0.001),ROS含量增加(P<0.001),细胞活性下降(P<0.001)。结论柿叶黄酮提取物对OGD/R损伤的HT22细胞氧化应激具有保护作用,其机制可能与激活Nrf2/HO-1信号通路有关。

关键词: 柿叶, 黄酮类物质, HT22细胞, 氧糖剥夺, 转录因子NF-E2相关因子, 信号通路

Abstract: Objective To investigate the protective effect of flavonoid components extracted from Diopyros Kaki(FLDK)on oxidative stress induced by oxygen glucose deprivation/reperfusion(OGD/R)in HT22 cells. Methods HT22 cells were randomly divided into normal control group(Control group), OGD/R group, OGD/R+FLDK treatment group(OGD/R+FLDK group), OGD/R +FLDK+transcription factor NF-E2 related factors(Nrf2)gene knockdowm group(OGD/R+FLDK+si-Nrf2 group). OGD/R model of HT22 cells were established by oxygen glucose deprivation for 3 h followed by a 24 h reperfusion; FLDK was added at the beginning of the OGD, and continued to the end of reperfusion; using si-Nrf2 to block the Nrf2 pathway. CCK-8 assay was used to detect cell viability; lactate deoxidizing enzyme(LDH)assay was used to detect the cell injury rate; related kits were used to detect the MDA content, 山东大学学报 (医学版)56卷6期 -崔春英,等.柿叶黄酮提取物对氧糖剥夺/复糖复氧损伤的HT22细胞氧化应激的保护作用 \=-SOD and GSH-Px activity; DCFH-DA fluorescent probe was used to detect the reactive oxygen species(ROS)levels; Western blotting was used to detect the nuclear-Nrf2 and HO-1 proteins expression. Results Compared with the control group, the cell activity was decreased(P<0.001); the cell injury rate was increased(P<0.001); the ROS and MDA content were increased(P<0.001); the antioxidant enzyme SOD and GSH-Px activity were decreased(P<0.001); HO-1 protein expression was increased(P=0.009)and the nuclear-Nrf2 protein expression was increased(P=0.016)in the OGD/R group. Compared with the OGD/R group, the cell activity was increased(P<0.001), the cell injury rate was decreased(P<0.001), the ROS and MDA content were decreased(P<0.001), the antioxidant enzyme SOD and GSH-Px activity were increased(P<0.001), the HO-1 protein expression was increased(P<0.001)and nuclear-Nrf2 protein expression was increased(P=0.005). Compared with the OGD/R+FLDK group, the HO-1 protein expression was decreased(P<0.001),the nuclear-Nrf2 protein was decreased(P=0.001), the ROS level was increased(P<0.001)and the cell activity was decreased(P<0.001). Conclusion FLDK can attenuate the oxidative stress in HT22 cells induced by OGD/R injury, and it may be associated with the activation of the Nrf2/HO-1 signaling pathway.

Key words: Diopyros Kaki, Flavonoid components, HT22 cells, Oxygen glucose deprivation, Nrf2, Signaling pathway

中图分类号:

R743

崔春英,申超,洪艳,陈建,刘雪平. 柿叶黄酮提取物对氧糖剥夺/复糖复氧损伤的HT22细胞氧化应激的保护作用[J]. 山东大学学报 (医学版), 2018, 56(6): 6-12.

CUI Chunying, SHEN Chao, HONG Yan, CHEN Jian, LIU Xueping. Protective effect of flavonoid components extracted from Diopyros Kaki on oxidative stress induced by OGD/R in HT22 cells[J]. Journal of Shandong University (Health Sciences), 2018, 56(6): 6-12.

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