依布硒林减轻DMT1诱导的铁死亡在实验性大鼠蛛网膜下腔出血中的研究

目的 观察大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后二价金属离子转运体(divalent metal transportor 1,DMT1)的表达变化及铁死亡情况,探讨依布硒林可能通过抑制DMT1的表达减轻铁死亡,从而对SAH后早期脑损伤起保护作用.方法 血管内穿刺法构建SD大鼠SAH模型,免疫荧光检测脑皮质中DMT1、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)表达定位,Western blot检测脑皮质中SAH后72 h内各时间点及侧脑室注射依布硒林后DMT1、GPX4的表达变化,检测细胞内铁沉积情况、铁含量、脂质过氧化物产物丙二醛(malonaldehyde,MDA)等铁死亡指标,以及大鼠脑水肿和神经功能学评分变化.结果 免疫荧光检测结果显示DMT1、GPX4表达于大鼠脑皮质神经元胞质内.Western blot检测结果显示,与假手术组相比,DMT1表达先升高,24 h降低后再次升高,48、72 h仍增高(P＜0.01);而SAH后GPX4的表达降低,24 h显著降低(P＜0.01),随后48、72 h继续降低(P＜0.01).与SAH组、溶剂组相比,依布硒林2 mg/kg组中DMT1降低(P＜0.01);依布硒林4 mg/kg干预后DMT1表达降低显著(P＜0.01),GPX4表达增高显著(P＜0.01).与SAH组、溶剂组相比,依布硒林4 mg/kg组中细胞内铁沉积减少,铁含量、MDA减少(P＜0.01);GSH含量、GPX4活性增加(P＜0.01).神经功能学评分、脑水肿得到改善(P＜0.01).结论 依布硒林可能通过抑制DMT1的表达减少铁向胞内转运继而减轻铁死亡,从而对SAH后早期脑损伤起保护作用.

Ebselen relieves ferroptosis induced by divalent mental transporter 1 in rats with subarachnoid hemorrhage

Objective To investigate the expression of divalent mental transporter 1 (DMT1) and ferroptosis in rats after subarachnoid hemorrhage (SAH) and explore the neuroprotective effect of ebselen in association of inhibiting DMT1 against early brain injury after SAH.MethodS Rat models of SAH were established by intravascular puncture.Immunofluorescence assay was used to identify the expression and location of DMT1 and glutathione peroxidase 4 (GPX4) in the brain cortex.Ebselen was administered by injection to the SAH.Western blotting was used to detect the expression of DMT and GPX4 at 72 h after the injury or after ebselen intervention.Intracellular iron deposition,iron content,and malonaldehyde (MDA) were measured to evaluate the ferroptosis.Neurological function and brain edema were observed to evaluate the early brain injury.Results Immunofluorescence assay indentified DMT1 and GPX4 were both expressed in the cytoplasm of neurons in the brain cortex.Western blot results revealed that the expression level of DMT1 was increased then decreased in 24 h,and kept rising in 48 and 72 h after SAH injury (P ＜ 0.01),while that of GPX4 was decreased at 24 h,and then further reduced in 48 and 72 h (P ＜0.01).Compared with the SAH group and vehicle group,injections of 1,2 and 4 mg/kg ebselen decreased the expression of DMT1 but increased that of GPX4,but significant differences were only found in the 2 and 4 mg/kg treatment groups (P ＜0.01).What's more,4 mg/kg ebselen also reduced iron deposition,iron and MDA contents (P ＜ 0.01),and elevated GSH content and GPX4 activity (P ＜0.01),and improved neurological function and augmented brain edema (P ＜ 0.01).Conclusion Ebselen may decrease the intracellular iron transport through inhibiting the expression of DMT1,then decrease the ferroptosis,and thus exert protective effect on the early brain injury in rats after SAH.