二氢杨梅素对铁过载导致的海马神经元细胞损伤的保护作用及其机制

Protective effect of dihydromyricetin against hippocampal neuronal injury caused by iron overload and its mechanism

目的探讨二氢杨梅素(DMY)对枸橼酸铁铵(FAC)导致的原代海马神经元细胞损伤的保护作用及其机制。方法取出生24 h SD乳鼠的原代海马神经元进行体外培养,使用CCK-8法检测不同浓度DMY处理后的原代海马神经元细胞的活性,确定DMY给药浓度。将原代海马神经元细胞分为对照组(H组)、100 mmol/L浓度的DMY处理组(I组)、250 mmol/L浓度的FAC处理组(J组)、100 mmol/L浓度的DMY与250 mmol/L浓度的FAC共处理组(K组),使用流式细胞术检测各组细胞中活性氧(ROS)的含量,使用比色法检测各组细胞中丙二醛(MDA)的含量,采用Western blot方法检测各组细胞中的NF-E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)和谷胱甘肽过氧化酶4(GPX4)蛋白的表达水平。结果CCK-8检测结果显示,在DMY浓度为100 mmol/L时,原代海马神经元细胞的存活率最高(F=9.95,P<0.05),故以该浓度用于后续实验。与H组相比,J组细胞中ROS和MDA含量明显升高(F=176.81、5523.35,P<0.05);与J组相比,K组细胞中ROS含量和MDA含量明显降低(F=18.21、412.96,P<0.05)。与H组相比,J组细胞中Nrf2、HO-1、GPX4蛋白相对表达量明显降低(F=27.35~81.32,P<0.05);与J组相比,K组细胞中的Nrf2、HO-1、GPX4蛋白相对表达量明显升高(F=8.74~21.46,P<0.05)。结论DMY可以缓解铁过载导致的原代海马神经元氧化应激损伤,其机制可能与其激活了原代海马神经元中的Nrf2-HO-1/GPX4通路有关。

Objective To investigate the protective effect of dihydromyricetin(DMY)against injury of primary hip-pocampal neurons caused by ferric ammonium citrate(FAC)and its mechanism.Methods Primary hippocampal neurons were collected from 24-hour neonatal Sprague-Dawley rats for in vitro culture,and CCK-8 assay was used to measure the viability of primary hippocampal neurons treated with different concentrations of DMY and determine the administration concentration of DMY.Primary hippocampal neurons were divided into control group(H group),100 mmol/L DMY treatment group(I group),250 mmol/L FAC treatment group(J group),and 100 mmol/L DMY+250 mmol/L FAC treatment group(K group).Flow cytometry was used to measure the content of reactive oxygen species(ROS)in each group;colorimetry was used to measure the content of malondialdehyde(MDA)in each group;Western blot was used to measure the protein expression levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and glutathione peroxidase 4(GPX4)in each group.Results CCK-8 assay showed the highest viability of primary hippocampal neurons at the concentration of 100 mmol/L for DMY(F=9.95,P<0.05),and therefore,this concentration was used for subsequent experiments.Compared with the H group,the J group had significant increases in the content of ROS and MDA(F=176.81,5523.35,P<0.05),and compared with the J group,the K group had significant reductions in the content of ROS and MDA(F=18.21,412.96,P<0.05).Compared with the H group,the J group had significant reductions in the relative protein expression levels of Nrf2,HO-1,and GPX4(F=27.35-81.32,P<0.05),and compared with the J group,the K group had significant increases in the relative protein expression levels of Nrf2,HO-1,and GPX4(F=8.74-21.46,P<0.05).Conclusion DMY can alleviate oxidative stress damage in primary hippocampal neurons due to iron overload,possibly by activating the Nrf2-HO-1/GPX 4 pathway in primary hippocampal neurons.