肠道微生物代谢产物对急慢性辐射损伤的救治作用及其机制研究

Radiation exposure in a mass casualty setting is a serious military and public health concern. Exposure to a high dose of irradiation is associated with bone marrow toxicity and gastrointestinal toxicity. Clinical evidence manifests the bone marrow and small intestine epithelium to be the major sites of injury during radiotherapy, owing to their higher sensitivity to ionizing radiation. Heretofore, radiation mediated toxicity remains a medical problem that urgently needs effective therapy. Our preliminary studies have demonstrated that fecal microbiota transplantation can be employed to fight against acute radiation syndrome. Hydrogen-water can alleviate total abdominal irradiation induced small intestinal toxicity through retaining the intestinal bacterial composition structure. On the basis of the previous data, we further assume that gut microbial metabolites might protect against acute and chronic injury induced by irradiation. To address the hypothesis, liquid chromatography will be performed to measure the metabolites of gut microbiota before and after radiotherapy. Protective function of metabolites against irradiation will be assessed by histological analysis, ELISA, flow cytometry, western blot, etc. Bacterial diversity analysis, transcriptome analyses and proteomoes analyses to uncover the underlying mechanism. We will employ gene-engineering mouse to further study the critical roles intestinal microbial metabolites in vivo. It is expected that the coming findings from the studies will provide theoretical and experimental evidence for enteric microbial metabolites as novel therapeutics to alleviate radiation induced injury. Therefore, the present study has of important significance and value in both the basic research of radiation toxicity and the clinical application in the future.

意外和医源性照射伴随着严重且不可逆的急慢性辐射损伤。急慢性辐射损伤的救治一直是放射医学领域的临床难题。肠道微生物在宿主生理病理进程中发挥着及其重要的作用。我们之前的研究证明粪菌移植能够救治急慢性辐射损伤。但是粪菌移植如何发挥功能及其作用机制尚不清楚。在申请人研究的基础上，本项目以肠道微生物代谢产物为抓手，建立全身照射和腹部局部照射两种实验模型，研究肠道微生物代谢产物对急慢性辐射损伤的救治作用。我们将运用流式细胞术、组学技术、ELISA等细胞与分子生物学技术在功能上明确肠道微生物代谢产物对于两种照射模型的救治作用。此外，运用转录组测序、基于质谱的蛋白组学分析、16sRNA测序、免疫印迹、实时定量PCR等分子生物学技术对肠道微生物代谢产物的作用机制进行深入的探讨，并运用基因工程鼠和人源永生细胞系对结果进行验证。项目的完成将阐明粪菌移植的作用机制，并为辐射防护与康复药物开发提供新思路和新靶点。

放射性肠损伤是意外核素照射和肿瘤放射治疗中常见的放射病，导致治疗提前终止，影响患者生活质量甚至危及生命。放射性肠损伤的机理尚待阐明，没有针对该放射病的临床用药。探究放射性肠损伤的机制，研发放射性肠损伤救治的新方法具有广泛而重要的临床应用前景和现实意义。项目负责人首次报道运用粪菌移植救治放射性肠损伤。而粪菌移植的临床应用伴随不同程度不良事件发生。经过筛选我们发现全身和局部照射后，小鼠粪便中戊酸、吲哚丙酸含量下降，粪菌移植后受体粪便中戊酸和吲哚丙酸含量明显回升。戊酸或吲哚丙酸单独使用均能够提高受照小鼠肠道完整性和功能，降低肠炎；提高受照小鼠生存率，缓解造血系统损伤，降低系统性炎症；重塑受照小鼠肠道微生态和小肠蛋白表达谱。机制研究显示戊酸提高小肠组织KRT1表达而实现放射性肠损伤的救治；吲哚丙酸激活小肠组织PXR/ACBP通路修复肠型放射损伤。通过水动力转染技术敲降小肠中KRT1，戊酸失去放射保护作用；敲降PXR或ACBP均可使吲哚丙酸放射保护作用消失。项目负责人的研究显示在医疗资源充足的情况下，可行粪菌移植救治放射损伤；当医疗资源紧张或患者不适于进行插管时，可口服肠道微生物代谢产物戊酸、吲哚丙酸来实现放射损伤的救治。我们的工作为肿瘤放射治疗病人康复以及核应急大规模人群救治提供了新的理论支撑和技术保障。此外，项目负责人针对放射损伤救治的两性差异展开研究。我们发现辛伐他汀能够缓解雄性放射性造血系统损伤和肠损伤，短期高脂饮食能够缓解雌性放射损伤；热量受限缓解雌性小鼠放射性肠炎，提高雄性小鼠肠道完整性；静注人免疫球蛋白能够修复雌性个体放射性造血和肠道损伤，而雄性个体需要补充肠道习居毛螺菌静注人免疫球蛋白才能发挥放射保护作用。这些工作首次证实了雌雄个体在放射损伤与救治中的差异，揭示放射损伤康复需考虑到患者性别。同时为核应急的大规模人群救治以及肿瘤放射治疗病人的康复提供了基于性别的“精准”康复策略和技术储备。