随着核技术的迅速发展，人们接触辐射的风险日益增加。无论核恐怖主义与核事故的威胁，还是临床上X射线诊断和肿瘤放射治疗等，都会对机体正常组织造成辐射损伤，因此，我们迫切需要找到一种有效的防护药物，来减轻电离辐射对机体正常组织的损害。到目前为止，氨磷汀（Amifostine）是唯一被FDA批准用于临床的小分子辐射防护剂，主要被用于减轻肿瘤患者接受放疗或者化疗时产生的副作用。但由于Amifostine存在一定的副作用和缺点制约了其在临床上的广泛应用。因此，开发新型辐射防护药物具有重要的临床意义和应用价值。本课题系统的研究了两种小分子化合物：其一对本课题组前期研发的新型辐射防护药物XH-008化合物进行了比较系统的研究，包括XH-008的小鼠30天存活率及DRF值，对辐射诱导造血系统、肺组织及肠损伤的防护作用以及发挥辐射防护作用的机制；其二对1,4-二硫苏糖醇（DTT）化合物在辐射诱导的急性造血系统及肠损伤中的防护作用进行了研究，并初步探究了其发挥辐射防护作用的机制。

第一部分：新型辐射防护药物XH-008对小鼠辐射损伤防护作用的研究

本文研究了新型辐射防护药物XH-008对电离辐射诱导造血系统、肺组织及肠损伤的防护作用，并探究了其发挥辐射防护作用的机制。30天存活率结果显示XH-008能显著提高不同照射剂量下（7.2 Gy、7.5 Gy、10 Gy、12.5 Gy、15 Gy全身辐射暴露，17 Gy右侧肺部辐射暴露和18 Gy腹部辐射暴露）小鼠的存活率，DRF值和Amifostine相近，药效和Amifostine相似。基于此，我们分别考察了XH-008对辐射诱导造血系统损伤、肺损伤以及肠损伤的防护作用。4 Gy全身辐射暴露结果显示XH-008能显著减轻受照小鼠外周血骨髓抑制和髓系偏斜，并能减少受照小鼠造血干细胞（HSCs）和造血祖细胞（HPCs）耗竭。17 Gy右侧肺部辐射暴露结果显示XH-008能显著减轻受照小鼠肺纤维化。15 Gy腹部辐射暴露结果显示XH-008能显著减轻受照小鼠小肠形态学损伤，并维持受照小鼠小肠隐窝细胞增殖和减少隐窝细胞凋亡。通过RNA测序，我们发现未照射小鼠经Amifostine给药共调控3746个基因变化，而XH-008只调控865个基因变化；照射前小鼠经Amifostine给药共诱导575个基因变化，照射前XH-008给药只诱导205个基因变化。KEGG通路数据库分析结果显示XH-008主要参与上调细胞内生物氧化、氨基酸合成与代谢通路，下调红细胞摄取氧和释放二氧化碳、清除血浆中亚铁血红素、激活NADPH氧化酶通路；Amifostine主要参与上调氨基酸合成与转换通路，以及下调胆固醇的生物合成、谷胱甘肽结合通路。之后，我们通过Real-time PCR和Western Blotting对上述结果进行验证。Real-time PCR结果显示XH-008能显著调控照射后隐窝细胞内氧化平衡、氨基酸合成与代谢相关基因的表达水平，Western Blotting结果显示XH-008能显著抑制受照小鼠小肠中NOX1蛋白表达。此外，通过流式细胞术，我们发现XH-008能显著抑制受照细胞lipid ROS高水平，表明XH-008能通过铁死亡通路发挥防护作用。GSH检测和Western Blotting结果显示XH-008能显著提高受照细胞GSH合成水平以及增加受照细胞和小鼠小肠隐窝中GPX4蛋白表达水平；细胞增殖实验显示XH-008显著改善电离辐射诱导的细胞增殖抑制；而且在腹部局部照射模型中，我们发现Liproxstatin-1能显著提高受照小鼠的存活率，表明XH-008能通过抑制辐射诱导铁死亡缓解机体损伤。最后，我们通过铁死亡激活剂进一步验证了XH-008对铁死亡的抑制作用，我们发现XH-008显著减轻铁死亡激活剂（CIL56, Erastin, IKE）诱导的细胞增殖抑制以及肠和肾损伤。综述所述，我们的研究表明，XH-008能显著减轻辐射诱导的小鼠造血系统、肺组织和肠损伤，其机制可能是通过调节氧化平衡、氨基酸合成与代谢及抑制辐射诱导的铁死亡实现。

第二部分：1,4-二硫苏糖醇（DTT）对小鼠急性辐射损伤防护作用的研究

本文探究了DTT对电离辐射诱导的急性造血系统、肠损伤的防护作用，并初步探讨了DTT发挥防护作用的机制。小鼠30天存活率表明，在全身致死剂量照射下，DTT能显著提高小鼠的存活率。之后，我们考察了DTT对辐射诱导的急性造血损伤和肠损伤的防护作用。在小鼠全身照射模型中，我们发现DTT给药能有效减轻受照小鼠骨髓抑制和髓系偏斜，以及增加受照小鼠造血干/祖细胞的自我更新和分化。在腹部局部照射模型中，我们发现DTT能有效缓解受照小鼠的体重下降和结肠长度缩短。免疫组化和免疫荧光结果显示，DTT能显著减少腹部局部照射小鼠小肠形态学损伤，并增加照射后小鼠小肠干细胞的存活和再生能力。此外，DTT还能减轻腹部局部照射诱导的小肠DNA损伤，并且减少受照小鼠小肠细胞凋亡。最后，我们运用Western Blotting探讨了腹部局部照射后小肠隐窝中促凋亡蛋白以及抗凋亡蛋白表达水平，结果显示DTT能显著抑制受照小鼠促凋亡p53, Bax, Bak蛋白表达以及增加抗凋亡Bcl-2蛋白表达。综上所述，我们的研究表明，DTT能有效缓解辐射诱导的小鼠急性造血系统和肠损伤，其部分机制可能是通过调节P53固有凋亡信号通路实现。

With the rapid development of nuclear technology, the threat of radiation exposure is increasing. Whether it’s the threat of terrorism and nuclear accidents, or that of X-ray diagnosis and radiotherapy in clinical, they will cause radiation injury to the normal tissues. Therefore, it is very important to find an effective prevention or treatment method to reduce ionizing radiation injury to the normal tissues. So far, amifostine is the only small molecule radioprotector approved by the FDA for clinical use. It is mainly used to reduce the injury to normal tissues for tumor patients after radiotherpy or chemotherapy. However, the side effects of amifostine severely limited its widespread application in clinical. Therefore, developing new radioprotectors has great clinical significance and application value. This subject studied two small molecule compounds systematically: One is the novel radioprotector XH-008 developed by the research group in the early stage has been systematically studied, including the 30-day survival rate and Dose Reduction Factor (DRF) of irradiated mice treated with XH-008, the protective effect of XH-008 on radiation-induced hematopoietic system, intestinal tissue and lung tissue injuries, and discussed its mechanism of being a radioprotector. It also studied the protective effect of 1,4-dithiothreitol (DTT) on radiation-induced acute hematopoietic system and intestinal tissue injuries, and discussed its mechanism of being a radioprotector preliminary.

Part I: Study on the protective effect of a new radioprotector XH-008 on radiation injury in mice

The current investigation was designed to explore the protective effects and mechanism of a new radioprotector XH-008 on ionizing radiation induced injuries. XH-008 treatment improved the 30-day survival rate of irradiated mice (mice were exposed to whole body irradiation (WBI) of 7.2 Gy, 7.5 Gy, 10 Gy, 12.5 Gy and 15 Gy, lung irradiation of 17 Gy and abdominal irradiation (ABI) of 18 Gy), the DRF of XH-008 and amifostine was 1.613 and 1.713 respectively. Then we explores the protective effect of XH-008 on hematopoietic injury, lung injury and intestinal injury induced by ionizing radiation. The results showed that XH-008 can significantly alleviated bone marrow suppression, myeloid skewing and hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) damage induced by WBI of 4 Gy. XH-008 also alleviated lung fibrous in the irradiated mice. XH-008 treatment protected mice from ABI-induced the morphological damage of the small intestine, maintain the proliferation of small intestinal crypt cells and reduce the apoptosis of small intestinal crypts after irradiation. RNA-sequence experiment showed that the non-irradiated mice were treated with amifostine, 3746 genes were regulated; in contrast, only 865 genes changed in XH-008 treated non-irradiated mice crypt cells 4 hours after XH-008 administration. When the irradiated mice treated with XH-008, 205 genes were regulated and amifostine may induce 575 gene varied. A reactome database enrichment analysis showed that XH-008 can up-regulated biological oxidations, amino acid synthesis and metabolic pathways and down-regulated erythrocytes take up oxygen and release carbon dioxide, scavenging of heme from plasma, and Rho GTPase active NADPH oxidases process; Amifostine can up-regulated amino acid synthesis and interconversion, down-regulated cholesterol biosynthesis and glutathione conjugation. Real-time PCR was verified the expression of pathways-related genes. Western blotting showed that XH-008 has greatly reduced the expression of NOX1 in the small intestine of mice. In addition, XH-008 significantly inhibited the level of lipid ROS in irradiated cells. GSH detection and western blotting showed that XH-008 significantly improved the synthesizing level of GSH in irradiated cells and increased the expression of CPX4 protein in irradiated cells and crypts in irradiated mice; as expected, XH-008 significantly reduced the irradiation-inhibited cell proliferation of IEC6 cells in vitro; and Liproxstatin-1 enormously improved the survival rate of irradiated mice, these results indicated that XH-008 alleviated irradiation induced injuries by inhibiting radiation-induced ferroptosis. Moreover, XH-008 significantly decreased ferroptosis activator-inhibited cell proliferation of IEC6 cells, and alleviated intestinal injury and kidney injury triggered by ferroptosis activator. In general, these results demonstrated that XH-008 can effectively alleviate hematopoietic, lung, and intestinal injuries in irradiated mice, the mechanism of which might be related to regulating the biological oxidations, amino acid synthesis and metabolism, and inhibiting radiation-induced ferroptosis.

Part II: Study on protective effect of 1,4-dithiothreitol(DTT) on acute radiation injury in mice

The current investigation was designed to evaluate the protective effects and mechanism of DTT as a radioprotector on ionizing radiation induced injuries. DTT treatment improved the survival of irradiated mice and ameliorated WBI-induced hematopoietic injury by attenuating myelosuppression and myeloid skewing, increasing self-renewal and differentiation of HPCs/HSCs. In addition, DTT treatment protected mice from ABI-induced changes in crypt-villus structures and function. Furthermore, treatment with DTT significantly enhanced the ABI-induced reduction in Olfm4 positive cells and offspring cells of Lgr5⁺ stem cells, including lysozyme⁺ Paneth cells and Ki67⁺ cells. Moreover, IR-induced DNA strand break damage, and the expression of proapoptotic-p53, Bax, Bak protein and antiapoptotic-Bcl-2 protein were reversed in DTT treated mice, and DTT also promoted small intestine repair after radiation exposure via the p53 intrinsic apoptotic pathway. In general, these results demonstrated the potential of DTT as effective protection against acute hematopoietic injury and intestinal injury after irradiation exposure, and that the mechanism which might be related to the regulation of P53-dependent intrinsic apoptotic signal.