Polydatin inhibits H<sub>2</sub>O<sub>2</sub>⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation

ZHU Ping; LI Jiaqi; YIN Mengchen; WANG Ruirui; LIU Baocheng; REN Guangwei

doi:10.16306/j.1008-861x.2023.04.004

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Polydatin inhibits H₂O₂⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation

Experiment Research | 更新时间：2023-08-04

- Polydatin inhibits H₂O₂⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 37, Issue 4, Pages: 28-35(2023)
- 作者机构：
  
  1.上海中医药大学体育部（上海　201203）
  2.上海中医药大学附属岳阳中西医结合医院肿瘤科（上海　200437）
  3.上海中医药大学附属龙华医院（上海　200032）
  4.上海中医药大学中医健康服务协同创新中心（上海　201203）
  5.上海市浦东新区三林社区卫生服务中心（上海　200124）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2023.04.004
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朱萍,李嘉旗,尹萌辰等.虎杖苷通过调控Keap1/Nrf2/HO⁃1抑制H₂O₂诱导的SH⁃SY5Y细胞氧化应激和线粒体损伤[J].上海中医药大学学报,2023,37(04):28-35.

ZHU Ping,LI Jiaqi,YIN Mengchen,et al.Polydatin inhibits H₂O₂⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2023,37(04):28-35.
朱萍,李嘉旗,尹萌辰等.虎杖苷通过调控Keap1/Nrf2/HO⁃1抑制H₂O₂诱导的SH⁃SY5Y细胞氧化应激和线粒体损伤[J].上海中医药大学学报,2023,37(04):28-35. DOI： 10.16306/j.1008-861x.2023.04.004.

ZHU Ping,LI Jiaqi,YIN Mengchen,et al.Polydatin inhibits H₂O₂⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2023,37(04):28-35. DOI： 10.16306/j.1008-861x.2023.04.004.

摘要

目的,2,探讨虎杖苷（PD）对由过氧化氢（H,2,O,2,）诱导的人神经母细胞瘤SH-SY5Y细胞损伤的影响及其作用机制。,方法,2,采用SH-SY5Y细胞进行实验，细胞用不同浓度的H,2,O,2,处理24 h，通过细胞计数试剂盒-8（CCK-8）法检测细胞活力，以确定H,2,O,2,的诱导浓度。PD预处理细胞2 h后，加入H,2,O,2,诱导22 h，CCK-8法检测细胞活力，以确定PD的浓度。将细胞分为二甲基亚砜（DMSO）组、H,2,O,2,组、H,2,O,2,+PD 20 μmol/L组和H,2,O,2,+PD 40 μmol/L组。膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）凋亡试剂盒检测细胞凋亡，流式细胞术测定细胞内总活性氧（ROS），酶联免疫吸附测定（ELISA）检测细胞中谷胱甘肽（GSH）、丙二醛（MDA）、超氧化物歧化酶（SOD）的含量，JC-1线粒体荧光探针检测线粒体膜电位，Western blot检测B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱氨酸蛋白酶-3（Caspase-3）、剪切的多腺苷二磷酸核糖聚合酶（Cleaved-PARP）、核因子E2相关因子2（Nrf2）、Kelch样环氧氯丙烷相关蛋白1（Keap1）和血红素氧合酶1（HO-1）蛋白的含量。,结果,2,①CCK-8实验结果显示，400 μmol/L浓度 H,2,O,2,处理后，SH-SY5Y细胞活力为60%，故选择400 μmol/L H,2,O,2,作为后续SH-SY5Y细胞的刺激条件；20、40、50、80 μmol/L PD可显著抑制H,2,O,2,诱导的SH-SY5Y细胞活力下降（,P<,0.05）。②Annexin V-FITC/PI凋亡实验结果显示，20、40 μmol/L PD能够抑制H,2,O,2,诱导的SH-SY5Y细胞凋亡（,P<,0.05）。Western blot结果显示，20、40 μmol/L PD能够抑制 H,2,O,2,诱导的SH-SY5Y细胞中凋亡相关蛋白Bax、Caspase-3表达（,P<,0.05），并促进抗凋亡蛋白Bcl-2的表达（,P<,0.05）。③20、40 μmol/L PD能减少H,2,O,2,诱导的SH-SY5Y细胞中ROS、MDA的含量（,P,<,0.05），以及增加GSH、SOD含量（,P,<,0.05）。④JC-1线粒体膜电位检测结果表明，20、40 μmol/L PD能够缓解H,2,O,2,诱导的SH-SY5Y细胞线粒体损伤。⑤Western blot结果显示，20、40 μmol/L PD能够显著抑制H,2,O,2,诱导的SH-SY5Y细胞中Keap1、Nrf2和HO-1蛋白水平的下调（,P<,0.05）。,结论,2,PD能够减少H,2,O,2,诱导的SH-SY5Y细胞凋亡、氧化应激以及线粒体损伤，其作用机制可能与激活Keap1/Nrf2/HO-1信号通路有关。

Abstract

Objective： To investigate the effect of polydatin （PD） on human neuroblastoma SH-SY5Y cell damage induced by hydrogen peroxide （H,2,O,2,） and its mechanism.,Methods,2,SH-SY5Y cells were treated with different concentrations of H,2,O,2, for 24 h， and cell viability was detected by cell counting kit-8（CCK-8） method to determine the induced concentration of H,2,O,2,. After 2 h of PD pretreatment， then induced with H,2,O,2, for 22 h， and the cell viability was detected by CCK-8 method to determine the concentration of PD. Cells were divided into DMSO group， H,2,O,2, group， H,2,O,2,+PD 20 μmol/L group and H,2,O,2,+PD 40 μmol/L group. Apoptosis was detected with annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） apoptosis kit. Total intracellular reactive oxygen species （ROS） were determined by flow cytometry. Glutathione peroxide （GSH）， malondialdehyde （MDA） and superoxide dismutase （SOD） in cells were detected by ELISA. Mitochondrial membrane potential was detected by JC-1 mitochondrial fluorescence probe. B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cysteine protein-3 （Caspase-3）， cleaved poly（adenosine diphosphate-ribose） polymerase （Cleaved-PARP）， nuclear factor E2 associated factor 2 （Nrf2）， Kelch-like epichlorohydrin associated protein 1 （Keap1） and heme oxygenase 1 （HO-1） protein contents were detected by Western blot.,Results,2,①CCK-8 experiment results showed that viability of SH-SY5Y cells was 60% after 400 μmol/L H,2,O,2, treatment， so 400 μmol/L H,2,O,2, was selected as the subsequent stimulation condition of SH-SY5Y cells； 20， 40， 50， 80 μmol/L PD could significantly inhibit the H,2,O,2,-induced viability decrease in SH-SY5Y cells （,P<,0.05）. ②Annexin V-FITC/PI apoptosis experiment results showed that 20， 40 μmol/L PD could inhibit SH-SY5Y cell apoptosis induced by H,2,O,2, （,P,<,0.05）. Western blot results showed that 20， 40 μmol/L PD could inhibit the expression of apoptosis-related proteins Bax and Caspase-3 in H,2,O,2,-induced SH-SY5Y cells （,P,<,0.05）， and promote the expression of anti-apoptosis protein Bcl-2 （,P,<,0.05）. ③20， 40 μmol/L PD could reduce H,2,O,2,-induced ROS， MDA contents， and increase GPH and SOD content in SH-SY5Y cells. ④The results of JC-1 mitochondrial membrane potential assay indicated that 20， 40 μmol/L PD could alleviate H,2,O,2,-induced mitochondrial damage in SH-SY5Y cells. ⑤Western blot results showed that 20， 40 μmol/L PD could significantly inhibit the down-regulation of Keap1， Nrf2 and HO-1 proteins in H,2,O,2,-induced SH-SY5Y cells （,P,<,0.05）.,Conclusion,2,PD can protect H,2,O,2,-induced apoptosis， oxidative stress， and mitochondrial damage in SH-SY5Y cells， and its mechanism may be related to the activation of Keap1/Nrf2/HO-1 signal pathway.

关键词

虎杖苷人神经母细胞瘤细胞氧化应激线粒体损伤过氧化物Keap1/Nrf2/HO-1信号通路

Keywords

polydatinhuman neuroblastoma cellsoxidative stressmitochondria damageperoxideKeap1/Nrf2/HO-1 signal pathway

references

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Polydatin inhibits H2O2⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation

DOI：10.16306/j.1008-861x.2023.04.004

摘要

Abstract

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Keywords

references

Polydatin inhibits H₂O₂⁃induced oxidative stress and mitochondrial damage in SH⁃SY5Y cells through Keap1/Nrf2/HO⁃1 regulation