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

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。

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

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.