摘要
目的:山楂叶黄酮是山楂叶中的药理活性成分,本研究探讨了山楂叶黄酮对H_(2)O_(2)诱导的PC12细胞损伤的保护作用及其机制。方法:通过H_(2)O_(2)诱导PC12细胞建立体外细胞模型,采用CCK-8法测定PC12细胞存活率,考察H_(2)O_(2)对PC12的细胞毒性及山楂叶黄酮对H_(2)O_(2)诱导PC12细胞损伤的保护作用;采用LDH法考察山楂叶黄酮对H_(2)O_(2)致PC12细胞毒性的影响;采用Hoechst 33342染色、AnnexinV-FITC/PI双染色法检测山楂叶黄酮对H_(2)O_(2)致PC12细胞凋亡的影响,JC-1法检测山楂叶黄酮对H_(2)O_(2)致PC12细胞线粒体膜电位的影响;采用探针DCFH-DA检测山楂叶黄酮对H_(2)O_(2)致PC12细胞内活性氧(ROS)的影响;采用超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)和丙二醛(MDA)试剂盒检测山楂叶黄酮对H_(2)O_(2)致PC12细胞内氧化状态的影响;采用Western Blot法检测PC12细胞内相关凋亡蛋白BAX、BCL-2和Caspase-3的表达以及核因子红细胞2相关因子2(Nrf-2)/ARE通路相关蛋白Nrf-2、NAD(P)H醌氧化还原酶1(NQO-1)、血红素加氧酶-1(HO-1)和Kelch样ECH相关蛋白1(Keap1)的表达。结果:与正常对照组比较,模型对照组PC12细胞存活率显著降低(P<0.01),细胞内ROS水平、细胞凋亡率显著升高(P<0.01),线粒体膜电位显著降低(P<0.01),SOD和CAT活性以及GSH含量显著降低(P<0.01),MDA含量显著升高(P<0.01),BAX和Caspase-3蛋白表达显著上调(P<0.05或P<0.01),BCL-2、Nrf-2、NQO-1和HO-1蛋白表达明显下调(P<0.05或P<0.01)。与模型对照组相比,山楂叶黄酮10μg/mL、20μg/mL组细胞存活率明显升高(P<0.05或P<0.01),细胞内ROS水平、细胞凋亡明显减少(P<0.05或P<0.01),线粒体膜电位显著升高(P<0.01),SOD和CAT活性以及GSH含量明显升高(P<0.05或P<0.01),MDA含量明显降低(P<0.05或P<0.01),BAX和Caspase-3蛋白表达明显下调(P<0.05或P<0.01),Bcl-2、Nrf-2、NQO-1和HO-1蛋白表达明显上调(P<0.05或P<0.01)。结论:山楂叶黄酮通过调节Nrf-2/ARE信号通路保护PC12细胞对抗H_(2)O_(2)诱导的氧化应激损伤和细胞凋亡。
Objective:Hawthorn leaf flavonoids have pharmacological activities. In this study, we explored the protective effect of hawthorn leaf flavonoids on H_(2)O_(2)-induced damage in PC12 cells and its mechanism. Methods: H_(2)O_(2) was used to treat PC12 cells to establish a vitro cell model. The CCK8 method was used to determine the viability of PC12 cells. The cytotoxicity of H_(2)O_(2) on PC12 cells and the protection of hawthorn leaf flavonoids on PC12 cells against H_(2)O_(2) were investigated. The LDH method was used to investigate the effects of hawthorn leaf flavonoids on H_(2)O_(2)-induced cytotoxicity of PC12 cells. Hoechst 33342 staining and AnnexinV-FITC/PI double staining methods were used to detect the effects of hawthorn leaf flavonoids on the H_(2)O_(2)-induced apoptosis of PC12 cells. The JC1 staining method was employed to determine the mitochondrial membrane potential of PC12 cells. Probe DCFH-DA was used to detect the H_(2)O_(2)-induced intracellular ROS in PC12 cells. Superoxide dismutase(SOD), catalase(CAT), glutathione(GSH), and malondialdehyde(MDA) assay kits were used to measure the oxidative stress of PC12 cells exposed to H_(2)O_(2). Western blotting was performed to test the expression of the apoptosis-associated proteins Bax, Bcl2, and Caspase-3 as well as the nuclear factor erythroid 2-related factor 2(Nrf2)/ARE pathway-associated proteins Nrf2, NAD(P)H quinone oxidoreductase 1(NQO1), heme oxygenase 1(HO1), and Kelch-like ECH-associated protein 1(Keap1) in PC12 cells. Results: Compared with the normal control group, the model group showed declined cell viability(P<0.01), elevated intracellular ROS level and apoptosis rate(P<0.01), decreased mitochondrial membrane potential, SOD activity, CAT activity, and GSH content(P<0.01), increased MDA content(P<0.01), up-regulated protein expression of Bax and Caspase-3(P<0.05 or P<0.01), while down-regulated protein expression of Bcl2, Nrf2, NQO1, and HO1(P<0.05 or P<0.01). Compared with the model group, 10 μg/mL and 20 μg/mL hawthorn leaf flavonoids enhanced cell viability(P<0.05 or P<0.01), reduced intracellular ROS level and apoptosis rate(P<0.05 or P<0.01), elevated mitochondrial membrane potential(P<0.01), increased SOD activity, CAT activity, and GSH content(P<0.05 or P<0.01), reduced MDA content(P<0.05 or P<0.01), down-regulated protein expression of Bax and Caspase-3(P<0.05 or P<0.01) while up-regulated protein expression of Bcl2, Nrf2, NQO1, and HO1(P<0.05 or P<0.01). Conclusion: Hawthorn leaf flavonoids protects PC12 cells from H_(2)O_(2)-induced oxidative stress and cell apoptosis through Nrf2/ARE signaling pathway.
作者
孙涛
邓婷
秦涛
谢丹妮
徐颖
Sun Tao;Deng Ting;Qin Tao;Xie Danni;Xu Ying(Key Laboratory of Standardization for Chinese Herbal Medicine,Ministry of Education,Key Laboratory of Distinctive Chinese Medicine Resources in Southwest China,Chengdu University of Traditional Chinese Medicine,Chengdu 611137;School of Medical Information Engineering,Chengdu University of Traditional Chinese Medicine,Chengdu 611137)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2021年第4期47-55,共9页
Pharmacology and Clinics of Chinese Materia Medica
基金
四川省科技厅应用基础研究项目(编号:2021YJ01780)
成都中医药大学“杏林学者”学科人才科研提升计划(编号:ZRQN2020008)。
