维生素D3对抗蛛网膜下腔出血后铁死亡减轻早期脑损伤的机制探索

Vitamin D3 alleviates early brain injury by combating ferroptosis after subarachnoid hemorrhage

摘要:

目的 研究维生素D₃（VitD₃）对抗蛛网膜下腔出血（SAH）后铁死亡减轻早期脑损伤（EBI）的效果。方法 实验分为体内和体外两部分，分别使用54只健康雄性SD大鼠和高分化PC12细胞。大鼠随机分为空白组、Vehicle组、VitD₃组，每组18只。采用血管内刺破法建立SAH模型。大鼠在造模前24 h接受VitD₃（100 ng/kg）或Vehicle腹腔注射，造模成功后2 h再次注射，空白组仅予麻醉处理。PC12细胞随机分为空白组、Hemin组、DMSO组、VitD₃组，使用氯化血红素(Hemin)模拟血红蛋白对神经元细胞的影响，DMSO组及VitD₃组在造模前24 h，使用含有DMSO（含量为0.01%）或VitD₃ (100 nmol/L)的完全培养基孵育，空白组及Hemin组仅做换液处理。24 h后去除原有培养基溶液，使用Hemin刺激PC12细胞2 h，DMSO组及VitD₃组，相应再次加入含原浓度DMSO或VitD₃的培养基液。在造模完成后24 h，酶联免疫吸附分析检测脑组织和PC12细胞中丙二醛(MDA)和活性氧(ROS)含量，Western blotting检测过氧化物酶体增殖物激活受体γ（PPARγ）、核因子红细胞2相关因子2（Nrf2）、谷胱甘肽过氧化物酶4（GPX4）蛋白的表达情况，并通过透射电镜观察线粒体形态学改变，Liperfluo检测PC12细胞脂质过氧化物（LPO）的量，流式细胞仪检测细胞凋亡情况。结果 与Vehicle组相比，VitD₃组大鼠MDA、ROS水平降低（P＜0.05），PPARγ、Nrf2、GPX4蛋白表达水平升高（P＜0.05）。与Hemin组相比，VitD₃组PC12细胞MDA、ROS水平降低，PPARγ、Nrf2、GPX4蛋白表达升高，LPO表达量降低，凋亡细胞数量减少(均P＜0.05）。结论 VitD₃能够在SAH后抑制铁死亡，是一种可靠的早期神经细胞保护剂。

Abstract:

Objective To investigate the role of vitamin D₃ （VitD₃） in alleviating early brain injury （EBI） by combating ferroptosis after subarachnoid hemorrhage （SAH）.Methods Both in vivo and in vitro experiments were conducted using 54 healthy male Sprague-Dawley rats and highly differentiated PC12 cells， respectively. The rats were randomly divided into blank group， Vehicle group， and VitD₃ group， with 18 rats in each group. The method of endovascular perforation was used to establish a model of SAH， and the rats were given intraperitoneal injection of VitD₃ （100 ng/kg） or Vehicle at 24 hours before modeling and at 2 hours after modeling， while the rats in the blank group were given anesthesia treatment alone. PC12 cells were randomly divided into blank group， Hemin group， DMSO group， and VitD₃ group， and Hemin was used to mimic the effect of hemoglobin on neuronal cells. 24 hours before modeling in the DMSO and VitD₃ groups， cells were incubated with complete medium containing DMSO （0.01%） or VitD₃ （100 nmol/L）. After 24 hours， the original medium was removed， and PC12 cells were stimulated with Hemin for 2 hours. Subsequently， the DMSO group and the VitD₃ group were supplemented with culture medium containing the original concentrations of DMSO or VitD₃， respectively. At 24 hours after modeling， ELISA was used to measure the levels of malondialdehyde （MDA） and reactive oxygen species （ROS） in brain tissue and PC12 cells； Western blotting was used to measure the protein expression levels of peroxisome proliferator-activated receptor gamma （PPARγ）， nuclear factor erythroid 2-related factor 2 （Nrf2）， and glutathione peroxidase 4 （GPX4）； transmission electron microscopy was used to observe mitochondrial morphological changes； Liperfluo was used to measure the level of lipid peroxide （LPO） in PC12 cells； flow cytometry was used to observe cell apoptosis.Results Compared with the Vehicle group， the VitD₃ group showed a decrease in MDA and ROS expression levels （P <0.05）， while the expression levels of PPAR γ， Nrf2， and GPX4 proteins increased （P <0.05）. Compared with the Hemin group， the VitD₃ group showed a decrease in MDA and ROS expression levels in PC12 cells， an increase in PPARγ， Nrf2， and GPX4 protein expression， a decrease in LPO expression， and a reduction in the number of apoptotic cells （all P <0.05）.Conclusions VitD₃ can inhibit ferroptosis after SAH， and it can be used as a reliable neuroprotective agent in early brain injury.