Huang Q, Ji D, Tian X, Ma L, Sun X
Department of Clinical Medicine, Biochemical Drugs Engineering and Technological Research Center of Anhui Province, Bengbu 233030, China.
Department of Pharmacy, Bengbu Medical College, Biochemical Drugs Engineering and Technological Research Center of Anhui Province, Bengbu 233030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):937-943. doi: 10.12122/j.issn.1673-4254.2022.06.19.
To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).
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, Fe 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.
Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells ( < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level ( < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells ( < 0.05) and lowered the levels of intracellular ROS and ferric iron content ( < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells ( < 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 ( < 0.05).
Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.
探讨小檗碱抑制小鼠海马神经元细胞（HT22）铁死亡的机制。
将培养的HT22细胞用30或60μmol/L小檗碱预处理2小时，然后暴露于0.5μmol/L厄洛替尼8小时，使用CCK-8、铁荧光探针、荧光染料（DAPI）和荧光探针（H2DCFH-DA）检测细胞增殖、细胞内三价铁水平、细胞内活性氧（ROS）变化和细胞凋亡。采用RT-qPCR和蛋白质印迹法检测细胞中Nrf2、HO-1和GPX4的mRNA和蛋白表达。我们进一步检测了用2μmol/L ML385（一种Nrf2抑制剂）、60μmol/L小檗碱和厄洛替尼处理细胞的效果，以探讨小檗碱对厄洛替尼诱导的神经元细胞铁死亡的保护机制。
用0.5μmol/L厄洛替尼处理显著降低了HT22细胞的活力（<0.05），并增加了ROS的产生、细胞凋亡率和三价铁水平（<0.05）。用30和60μmol/L小檗碱预处理均显著提高了厄洛替尼处理细胞的活力（<0.05），并降低了细胞内ROS水平和三价铁含量（<0.05）。RT-qPCR和蛋白质印迹法显示，小檗碱明显促进了细胞中Nrf2、HO-1和GPX4的表达（<0.05），而用ML385处理显著抑制了Nrf2-HO-1/GPX4通路，增加了细胞内ROS和三价铁含量，并减轻了小檗碱对厄洛替尼诱导的铁死亡的保护作用（<0.05）。
小檗碱可能通过激活Nrf2-HO-1/GPX4通路抑制厄洛替尼诱导的HT22细胞铁死亡。
