维生素D3通过对抗蛛网膜下腔出血后铁死亡减轻早期脑损伤

Vitamin D3 Alleviates Early Brain Injury by Combating Ferroptosis after Subarachnoid Hemorrhage

摘要:

目的 探索维生素D₃(vitamin D₃,VitD₃)通过对抗蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后铁死亡对早期脑损伤(early brain injury,EBI)的影响。方法 体内实验采用54只健康雄性SD大鼠,随机分为三组：空白组、SAH+Vehicle组、SAH+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小时接受VitD(100ng/kg)或Vehicle腹腔注射,造模成功后2小时再次注射。24小时后,Elisa检测脑组织中丙二醛(malondialdehyde,MDA)和活性氧(reactive oxygen species,ROS)含量,Western blot检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-γ,PPARγ)、核因子红细胞2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白的表达,并通过透射电镜观察线粒体形态学改变。体外实验采用PC12细胞作为研究对象随机分为四组：空白组、Hemin组、Hemin+DMSO组、Hemin+VitD₃组。使用Hemin模拟血红蛋白对神经元细胞的影响。PC12细胞在造模前24小时使用含有VitD₃(100umol/L)或DMSO(含量＜0.01%)的完全培养基孵育,造模后2小时再次加入并孵育。造模后24小时,使用Elisa检测PC12细胞中丙二醛(malondialdehyde,MDA)和活性氧(reactive oxygen species,ROS)含量,Western blot检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor-γ,PPARγ)、核因子红细胞2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白的表达,并通过透射电镜观察线粒体形态学改变,Liperfluo检测脂质过氧化物(LPO)的量,流式细胞仪检测细胞凋亡情况。结果 VitD₃预处理可以降低MDA、ROS水平(＜0.05),蛋白水平层面可观察到VitD₃预处理后PPARγ、Nrf2、GPX4蛋白表达较对照组明显升高(＜0.05),LPO表达量相较于对照组明显降低(＜0.05),流式检测预给予VitD₃后凋亡细胞数量减少(＜0.05)。结论 VitD₃可以在SAH后抑制铁死亡,是一个可靠的早期神经细胞保护剂。

Abstract:

Objective To explore the effect of vitamin D₃(VitD₃) on early brain injury (EBI) by combating ferroptosis after subarachnoid hemorrhage (SAH). Method: 54 healthy male SD rats were randomly divided into three groups for in vivo experiments: blank group, SAH+vehicle group, and SAH+VitD₃ group, with 18 rats in each group. Establish SAH model using intravascular puncture method. Rats received intraperitoneal injection of VitD₃ (100ng/Kg) or Vehicle 24 hours before modeling, and were injected again 2 hours after successful modeling. 24 hours later, Elisa detected the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in brain tissue. Western blot was used to detect the expression of peroxisome proliferator activated receptor - γ (PPAR γ), nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4) proteins, and mitochondrial morphological changes were observed by transmission electron microscopy. In vitro experiments used PC12 cells as research subjects and randomly divided them into four groups: blank group, hemin group, hemin+DMSO group, and hemin+VitD₃ group. Simulate the effect of hemoglobin on neuronal cells using hemin. PC12 cells were incubated in complete culture medium containing VitD₃ (10umol/L) or DMSO (content<0.01%) 24 hours before modeling, and then added and incubated again 2 hours after modeling. 24 hours after modeling, Elisa was used to detect the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in PC12 cells, Western blot was used to detect the expression of peroxisome proliferator activated receptor - γ (PPAR γ), nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4) proteins, and transmission electron microscopy was used to observe mitochondrial morphological changes. Lipid peroxidation was used to detect the amount of lipid peroxides (LPO). Reuslts: VitD₃pretreatment can reduce MDA and ROS levels (<0.05). At the protein level, it can be observed that the expression of PPAR γ, Nrf2, and GPX4 proteins is significantly increased after VitD₃ pretreatment compared to the control group (<0.05), and the expression of LPO is significantly decreased compared to the control group (<0.05), the number of apoptotic cells decreased after pre-administration of VitD₃ as detected by flow cytometry ( < 0.05). Conclusion: VitD₃ can inhibit ferroptosis after SAH and is a reliable early neuroprotective agent.