Mitigation of total-body irradiation (TBI) in C57BL/6 mice by two drugs, which target apoptosis and necroptosis respectively, increases survival compared to one drug alone. Here we investigated whether the biomarker (signature)directed addition of a third anti-ferroptosis drug further mitigated TBI effects. C57BL/6NTac female mice (30-33 g) received 9.25 Gy TBI, and 24 h or later received JP4-039 (20 mg/kg), necrostatin-1 (1.65 mg/kg) and/or lipoxygenase-15 inhibitor (baicalein) (50 mg/kg) in single-, dual- or three-drug regimens. Some animals were sacrificed at days 0, 1, 2, 3, 4 or 7 postirradiation, while the majority in each group were maintained beyond 30 days. For those mice sacrificed at the early time points, femur bone marrow, intestine (ileum), lung and blood plasma were collected and analyzed for radiation-induced and mitigator-modified levels of 33 pro-inflammatory and stress response proteins. Each single mitigator administered [JP4-039 (24 h), necrostatin-1 (48 h) or baicalein (24 h)] improved survival at day 30 after TBI to 25% (P = 0.0432, 0.2816 or 0.1120, respectively) compared to 5% survival of 9.25 Gy TBI controls. Mice were administered the drug individually based on weight (mg/kg). Drug vehicles comprised 30% cyclodextrin for JP4-039 and baicalein, and 10% Cremphor-EL/10% ethanol/80% water for necrostatin-1; thus, dual-vehicle controls were also tested. The dual-drug combinations further enhanced survival: necrostatin-1 (delayed to 72 h) with baicalein 40% (P = 0.0359); JP4-039 with necrostatin-1 50% (P = 0.0062); and JP4-039 with baicalein 60% (P = 0.0064). The three-drug regimen, timed to signature directed evidence of onset after TBI of each death pathway in marrow and intestine, further increased the 30-day survival to 75% (P = 0.0002), and there was optimal normalization to preirradiation levels of inflammatory cytokine and stress response protein levels in plasma, intestine and marrow. In contrast, lung protein levels were minimally altered by 9.25 Gy TBI or mitigators over 7 days. Significantly, elevated intestinal proteins at day 7 after TBI were reduced by necrostatin-1-containing regimens; however, normalization of plasma protein levels at day 7 required the addition of JP4-039 and baicalein. These findings indicate that mitigator targeting to three distinct cell death pathways increases survival after TBI.

中文翻译：

---

抗铁死亡药物通过阻止细胞凋亡和坏死性凋亡的药物增强全身辐射缓解。


与单独使用一种药物相比，分别针对细胞凋亡和坏死性凋亡的两种药物可减轻 C57BL/6 小鼠的全身照射 (TBI)，从而提高了存活率。在这里，我们研究了生物标志物（特征）是否指导添加第三种抗铁死亡药物进一步减轻了 TBI 的影响。 C57BL/6NTac 雌性小鼠 (30-33 g) 接受 9.25 Gy TBI，24 小时或之后接受 JP4-039 (20 mg/kg)、necrostatin-1 (1.65 mg/kg) 和/或脂氧合酶 15 抑制剂（黄芩素） ) (50 mg/kg) 单药、双药或三药治疗方案。一些动物在照射后第0、1、2、3、4或7天处死，而每组中的大多数动物维持超过30天。对于那些在早期时间点处死的小鼠，收集股骨骨髓、肠（回肠）、肺和血浆并分析 33 种促炎和应激反应蛋白的辐射诱导和缓解修饰水平。与对照组相比，给予每种单一缓解剂 [JP4-039（24 小时）、necrostatin-1（48 小时）或黄芩素（24 小时）] 将 TBI 后第 30 天的生存率提高至 25%（分别为 P = 0.0432、0.2816 或 0.1120） 9.25 Gy TBI 对照的存活率为 5%。根据体重（mg/kg）对小鼠单独给药。 JP4-039 和黄芩素的药物载体为 30% 环糊精，necrostatin-1 的药物载体为 10% Cremphor-EL/10% 乙醇/80% 水；因此，还测试了双车辆控制。双药组合进一步提高了生存率：necrostatin-1（延迟至 72 小时）与黄芩素 40%（P = 0.0359）； JP4-039 含 necrostatin-1 50% (P = 0.0062)；和含有黄芩素 60% 的 JP4-039 (P = 0.0064)。三种药物方案，根据 TBI 后骨髓和肠道各死亡途径的直接发病证据进行定时，进一步将 30 天生存率提高至 75%（P = 0.0002），血浆、肠道和骨髓中炎症细胞因子和应激反应蛋白水平的辐射前水平达到最佳正常化。相比之下，7天内9.25 Gy TBI或缓解剂对肺蛋白水平的影响很小。值得注意的是，含有 necrostatin-1 的治疗方案可降低 TBI 后第 7 天升高的肠道蛋白水平。然而，第 7 天血浆蛋白水平正常化需要添加 JP4-039 和黄芩素。这些发现表明，针对三种不同细胞死亡途径的缓解剂可提高 TBI 后的存活率。