芍药汤调控Tuft细胞促Dclk1表达加速隐窝修复干预放射性肠损伤的生物学效应研究

Radiation-induced DNA double-strand breaks lead to structural destruction of intestinal crypts is the key to radiation-induced intestinal lesions. ATM-activated DDR directly participates in the crypt structure repair process. Recent studies have shown that Tuft cells promote Dclk1 expression to accelerate crypt repair. It has been confirmed clinically that Shaoyao Tang, which is characterized by treating incontinent syndrome with dredging method, is effective in interfering with radiation-induced intestinal injury. Probably its use of heat and dampness is directed against the radiation heat poison effect, and the work of reconciling regulation of qi and blood is compatible with the biomechanical behavior of crypt repair. According to this research hypothesis:Shaoyao Tang can accelerate the ATM activation of DDR-induced downstream cascades by modulating the expression of Dclk1 in Tuft cells, competes for cell apoptosis, and interferes with radioactive intestinal damage crypt repair. This project intends to construct a classic animal model of radiation-induced intestinal injury, and to pass immunohistochemistry, ELISA, WB, etc. To analyze the apoptosis-proliferation and expression of related cytokines in crypt cells to reveal the key target of crypt repair of radioactive intestinal lesions, explain the pathophysiology of traditional Chinese medicine for radiation-induced intestinal lesions, and enrich the treatment methods and prescriptions for TCM tumors.

放射线引起的DNA双链断裂导致肠道隐窝结构破坏是放射性肠损伤病变的关键。ATM激活DDR直接参与隐窝结构修复过程，最新研究表明Tuft细胞促Dclk1表达加速隐窝修复。临床证实体现通因通用治法特点的芍药汤干预放射性肠损伤疗效确切，盖其清热燥湿之用针对了放射线热毒效应，调和气血之功则契合了隐窝修复的生物力学行为。据此提出研究假说：芍药汤能通过调控Tuft细胞促Dclk1表达加速ATM激活DDR诱发下游级联反应，竞争细胞凋亡，干预放射性肠损伤隐窝修复。本项目拟构建放射性肠损伤经典动物模型，通过免疫组织化学法、ELISA、WB等检测技术，测析隐窝细胞凋亡-增殖、相关细胞因子表达，以揭示放射性肠损伤隐窝修复关键靶点，阐释放射性肠损伤的中医病理生理，丰富中医肿瘤的治法与方剂。

背景：芍药汤具有调和气血的作用，前期临床研究发现：芍药汤能降低急性放射性肠炎患者的发生率，改善临床症状，减轻粘膜损伤，加快肠道损伤修复，但其具体机制不详。内容：1.观察芍药汤对肠道隐窝的保护作用及对肠粘膜基质胶原纤维沉积的抑制作用；2.观察芍药汤对氧化及炎症的作用效应；3.观察芍药汤对结肠组织细胞凋亡的抑制作用；4.探究芍药汤抑制结肠粘膜损伤细胞凋亡的具体机制；5.观察芍药汤对结肠黏膜屏障相关机制的影响；6.体外实验观察芍药汤对IEC-6细胞凋亡的抑制作用及机制。结果：1.芍药汤高剂量组见轻微粘膜上皮细胞坏死脱落，隐窝未见明显受损；芍药汤小鼠结肠组织胶原纤维数量明显低于模型组；2.芍药汤高剂量组MDA的表达明显受到抑制，而SOD的表达明显增加。同时，芍药汤高剂量组COX、LPS、IL-6、IL-1β、TNF-α促炎因子水平明显下降；3.芍药汤组阳性TUNEL免疫荧光信号随剂量依赖性方式显著减弱；4.芍药汤高剂量组ATM、Dclk-1、MRE-11、Bcl-2显著升高，Bax、Caspase-3、Cyto-c显著降低；5.芍药汤高剂量组治疗后P53含量降低，Claudin-1表达增加；6.细胞实验证实芍药汤组Bax、Cyto-c和Caspase-3表达降低，同时Bcl-2表达升高， IEC-6细胞线粒体凋亡受到抑制。结论：1.芍药汤高剂量组抑制小鼠放射性肠炎效果最优，芍药汤具有保护肠道隐窝，减轻肠粘膜纤维化的作用；2.芍药汤可能通过抑制C57BL/6小鼠结肠组织中的氧化应激和炎症损伤达到缓解RE的作用，具体表现为下调MDA、COX、LPS、IL-6、IL-1β和TNF-α的表达，上调T-SOD的生成。3.芍药汤通过增强Dclk-1和Claudin-1的蛋白表达，进而促进ATM/MRE-11的表达，同时限制p53蛋白表达，来维持肠道屏障功能。4.芍药汤可能会通过抑制Bax、Cyto-c和Caspase-3，同时提高Bcl-2蛋白表达，从而阻断隐窝细胞线粒体凋亡。5.芍药汤改善放射性肠炎的机制可能与抑制炎症和凋亡有关。意义：本项目揭示了芍药汤促隐窝修复干预放射性肠损伤的生物学机制，为临床应用芍药汤治疗放射性肠炎提供基础研究支持，同时阐释放射性损伤的中医病理生理，加深了中医对放射性肠损伤的认识。