Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia-ischemia

These data present a study seeking to assess the effects of increasing 7-DHC concentrations for neuroprotection in cell culture and an in vivo neonatal (P9) mouse model of unilateral carotid artery ligation followed by hypoxia (30 min at 8%). The tabs correlate to each of the figures in the manuscript: Tab "fig 1B": shows RSL-3 mediated ferroptosis in HT1080 cells pre-treated for 24 h with vehicle (VEH), 50 nM cariprazine (CAR), 500 nM aripiprazole (ARI), 500 nM trazodone (TRZ) or 50 nM lovastatin (LOV). Cell viability was assessed with Alamar blue. CAR, ARI and TRZ are potent DHCR7-inhibiting medications and significantly suppress ferroptotic induced cell death. LOV is a potent HMGCR inhibitor and it does not prevent ferroptosis, indicating that the ferroptosis suppression is not due to cholesterol deprivation, but to an increase in 7-DHC. Tab "fig 2B": shows HT1080 cells pre-treated with 1, 10 and 25 nM CAR for 24h before ferroptosis was induced with 25 nM RSL-3. Cells were incubated with RSL-3 for 24 h and cell viability was assessed with Alamar blue. Cell viability is significantly increased with CAR pre-treatment. Also shows HT1080 cells treated simultaneously with 25 nM RSL-3 and 1, 10 or 25 nM CAR for 24h (co-treatment with CAR and RSL-3). Cell viability was assessed with Alamar Blue. Tab "fig 3B": shows the effects of CAR on neuronal viability after 4 h of oxygen glucose deprivation (OGD). For the pre-treatment experiments, neurons were treated with vehicle, 1 nM, 10 nM or 25 nM CAR for 48 h before OGD. For the post-treatment experiments, neurons were treated with vehicle, 1 nM, 10 nM or 25 nM 30 min after OGD. Cell viability was assessed 24 h after the termination of OGD and were calculated by dividing the numbers of live neurons after OGD by the number of live neurons of the control (not exposed to OGD). 7-DHC levels were assessed by LC-MS/MS at the endpoint of the experiment. Concentrations of 7-DHC are elevated by CAR administration but consumed by OGD. Tab "fig 4": 7-DHC derived oxysterols are increased in the brain tissue of CAR-treated animals after hypoxic-ischemic brain injury (HIBI). Animals were injected with 0.2 mg/kg of CAR as described in Figure 5A. 7-DHC-derived oxysterols DHCEO and ep7DHC were not detected (NC) in the Sham+vehicle or HIBI+vehicle groups but were detected in both CAR groups. Tab "fig 5B-E": For the pre-treatment experiments, postnatal day 7 (P7) mice were injected with vehicle (VEH) or 0.2 mg/kg CAR 48 h before HIBI. For the post-treatment experiments, mice were injected with vehicle or 0.2 mg/kg CAR 30 min after HIBI. MDA levels assessed by TBARS are decreased in animals treated with CAR, indicating that CAR treatment decreases markers of phospholipid oxidation. Tissue viability assessed by 2,3,5-Triphenyltetrazolium Chloride (TTC) reveals increased tissue viability in HIBI animals treated with CAR.

本数据集配套一项研究，旨在评估提升7-脱氢胆固醇（7-DHC）浓度对细胞培养模型及单侧颈动脉结扎联合低氧（8%氧浓度，持续30分钟）的新生（出生后第9天，P9）小鼠体内缺血缺氧模型的神经保护作用。各表格对应手稿中的每一幅图片：
表格"fig 1B"：展示经溶剂（VEH）、50 nM卡利拉嗪（CAR）、500 nM阿立哌唑（ARI）、500 nM曲唑酮（TRZ）或50 nM洛伐他汀（LOV）预处理24小时的HT1080细胞中，RSL-3诱导的铁死亡现象。采用阿尔玛蓝（Alamar Blue）检测细胞活力。结果显示，卡利拉嗪、阿立哌唑与曲唑酮均为强效7-脱氢胆固醇还原酶（DHCR7）抑制剂，可显著抑制铁死亡诱导的细胞死亡；洛伐他汀为强效3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）抑制剂，无法阻止铁死亡，提示铁死亡的抑制并非源于胆固醇剥夺，而是源于7-DHC浓度升高。
表格"fig 2B"：展示提前24小时以1、10、25 nM卡利拉嗪预处理HT1080细胞，随后用25 nM RSL-3诱导铁死亡，继续孵育24小时后通过阿尔玛蓝检测细胞活力的结果，可见卡利拉嗪预处理可显著提升细胞活力。该表格同时展示了同时用25 nM RSL-3与1、10或25 nM卡利拉嗪共处理24小时的细胞组，同样通过阿尔玛蓝检测细胞活力。
表格"fig 3B"：展示卡利拉嗪对氧糖剥夺（OGD）4小时后神经元活力的影响。预处理实验中，神经元提前48小时分别经溶剂、1 nM、10 nM或25 nM卡利拉嗪处理后再行OGD；后处理实验中，神经元于OGD结束后30分钟分别经溶剂、1 nM、10 nM或25 nM卡利拉嗪处理。于OGD终止24小时后检测细胞活力，以OGD后活神经元数与未暴露于OGD的对照组活神经元数的比值计算相对活力。实验终点通过液相色谱-串联质谱（LC-MS/MS）检测7-DHC水平：卡利拉嗪给药可升高7-DHC浓度，而OGD会消耗7-DHC。
表格"fig 4"：展示缺血缺氧性脑损伤（HIBI）模型中，经卡利拉嗪处理的动物脑组织内7-DHC衍生氧固醇水平升高。实验动物按图5A所述方法注射0.2 mg/kg卡利拉嗪。Sham+溶剂组与HIBI+溶剂组中未检测到7-DHC衍生氧固醇DHCEO与ep7DHC（记为NC），而两个卡利拉嗪处理组均检测到上述两种氧固醇。
表格"fig 5B-E"：预处理实验中，出生后第7天（P7）小鼠于HIBI造模前48小时注射溶剂或0.2 mg/kg卡利拉嗪；后处理实验中，小鼠于HIBI造模后30分钟注射溶剂或0.2 mg/kg卡利拉嗪。硫代巴比妥酸反应物（TBARS）法检测的丙二醛（MDA）水平在卡利拉嗪处理的动物中降低，提示卡利拉嗪治疗可降低磷脂氧化标志物水平。通过2,3,5-三苯基氯化四氮唑（TTC）检测组织活力可见，卡利拉嗪处理的HIBI动物脑组织存活面积显著提升。