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

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

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+30μmol/L BBR组、Erastin+60μmol/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60μmol 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+60μmol/L BBR组、Erastin+60μmol/L BBR+2μmol 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0.5μmol/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+30μmol/L BBR组和Erastin+60μmol/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。

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 60μmol/L berberine for 2 h before exposure to 0.5μmol/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 2μmol/L ML385(a Nrf2 inhibitor),60μmol/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 0.5μmol/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 60μmol/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.