南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (6): 937-943.doi: 10.12122/j.issn.1673-4254.2022.06.19

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小檗碱通过激活 Nrf2-HO-1/GPX4 通路抑制小鼠海马神经元HT22细胞的铁死亡

黄庆洋，纪东东，田绣云，马琳艳，孙小锦   

  1. 蚌埠医学院临床医学院；蚌埠医学院药学院，安徽省生化药物工程技术研究中心，安徽 蚌埠 233030
  • 出版日期:2022-06-20 发布日期:2022-06-27

Berberine inhibits erastin-induced ferroptosis of mouse hippocampal neuronal cells possibly by activating the Nrf2-HO-1/GPX4 pathway

HUANG Qingyang, JI Dongdong, TIAN Xiuyun, MA Linyan, SUN Xiaojin   

  1. Department of Clinical Medicine, Department of Pharmacy, Bengbu Medical College, Biochemical Drugs Engineering and Technological Research Center of Anhui Province, Bengbu 233030, China
  • Online:2022-06-20 Published:2022-06-27

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摘要： 目的 探讨小檗碱对于Erastin诱导小鼠海马神经元HT22细胞的铁死亡的保护作用及其可能机制。方法 以HT22小鼠海马神经元细胞为研究对象，分为对照组、Erastin模型组、Erastin+30 μmol/L BBR组、Erastin+60 μmol/L BBR组。采用CCK-8法、特异性 Fe2+ 荧光探针、荧光染料（DAPI）检测和荧光探针（H2DCFH-DA）检测各实验组细胞的增殖情况、活性铁水平、细胞凋亡和活性氧（ROS）变化。 RT-qPCR和Western blot分别检测各实验组细胞的Nrf2、HO-1、GPX4 mRNA和蛋白表达情况。以60 μmol BBR的最适浓度来进一步探究其作用机制，分为对照组、Erastin模型组、Erastin+60 μmol/L BBR组、Erastin+60 μmol/LBBR+2 μmol Nrf2抑制剂 ML385组。通过使用荧光探针和Western blot检测Nrf2抑制剂（ML385）作用后的活性铁的水平、活性氧含量以及Nrf2、HO-1、GPX4蛋白的表达来验证小檗碱调节的Nrf2-HO-1/GPX4通路对Erastin处理的HT22细胞的保护作用。结果 0.5 μmol/L Erastin作用于HT22细胞8 h，细胞存活率与对照组相比显著被抑制（P<0.05）；同时细胞凋亡、ROS以及活性铁含量增加（P<0.05）。与Erastin组比较，Erastin+30 μmol/L BBR组和Erastin+60 μmol/L BBR组的细胞存活率明显升高（P<0.05），同时显著降低细胞凋亡、ROS以及活性铁含量（P<0.05）。小檗碱增加 HT22细胞中Nrf2、HO-1、GPX4基因及蛋白的 表达量（P<0.05）。加入Nrf2抑制剂ML385后，Nrf2-HO-1/GPX4通路被抑制，并且ROS以及活性铁含量升高（P<0.05）。结论 Erastin诱导HT22细胞发生铁死亡，小檗碱抑制Erastin诱导的铁死亡，可能机制是激活了Nrf2-HO-1/GPX4通路。

关键词: 小檗碱；Erastin；HT22细胞；铁死亡；神经保护；Nrf2-HO-1/GPX4

Abstract: Objective To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22). Methods Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2 + fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells. Results Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P<0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P<0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P<0.05) and lowered the levels of intracellular ROS and ferric iron content (P<0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P<0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P<0.05). Conclusion Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/GPX4 pathway.

Key words: berberine; erastin; HT22 cells; ferroptosis; neuroprotection; Nrf2-HO-1/GPX4