【作者】 顾佳；

【导师】 徐加英；

【作者基本信息】 苏州大学 ， 放射医学， 2022， 硕士

【摘要】 目的:放射性肠损伤(radiation-induced intestinal injury,RⅢ)是由辐射事故或者盆腹腔及腹膜后肿瘤患者接受放射治疗后引起的肠道主要并发症。目前针对RⅢ发病机制的研究主要包括:机械屏障、化学屏障、免疫屏障和生物屏障损伤等。辐射导致肠道结构受损,引发肠道吸收和保护功能障碍,临床表现为恶心、呕吐、腹痛、腹泻、出血、肠穿孔等,患者甚至可因感染致休克或死亡。因此,RⅢ严重影响患者的生活质量和临床肿瘤的疗效,现尚无有效治疗手段。近年来,肠道菌群和疾病的发生发展已成为研究热点。辐射引起的肠道微环境失调引发了肠功能紊乱。有研究表明,肠道菌群及其代谢产物的改变可重塑肠稳态,改善RⅢ。因此,能够调节肠道菌群的RⅢ防护剂,已成为放射医学领域的研究热点。血根碱(sanguinarine,SAN)是一种具有抗菌、抗炎和抗肿瘤等功效的天然小分子化合物,目前是作为药物饲料添加剂广泛使用于养殖业。本课题组前期研究发现血根碱对辐射引起的小鼠急性器官损伤有预防作用,且对肠组织更为明显,但具体机制尚不清楚。本研究从体外和体内实验系统研究血根碱对RⅢ的防护作用及其机制,以期为临床RⅢ的防护提供新的选择。方法:(1)体外实验:为了进一步验证在细胞水平上,血根碱对放射性损伤的作用及机制,采用了CCK-8法检测了不同浓度的血根碱对小鼠肠上皮细胞系-6(IEC-6)的毒性;X射线照射前用血根碱预处理30分钟,分析在不同处理条件下IEC-6细胞活力和克隆形成能力的改变;Western blot法检测细胞凋亡相关蛋白NF-κB、Caspase-8和Caspase-3表达水平的改变,流式细胞术检测凋亡细胞数目的变化。(2)体内实验:6-8周龄C57BL/6J小鼠,适应性喂养1周后,随机为对照组(Con)、腹部照射组(whole abdominal irradiation,WAI)和血根碱+腹部照射(SAN+WAI)联合组,采用10 Gy的X射线腹部照射小鼠,建立RⅢ动物模型。于照射前3天及照射前30分钟给予2.5 mg/kg的血根碱或等体积生理盐水腹腔注射。为了研究造血系统损伤的情况,H&E染色法用于检测小鼠脾脏损伤、骨髓细胞微核试验分析骨髓细胞染色体损伤、血常规检测分析外周血细胞计数的变化。为了明确肠道系统的损伤情况,H&E染色和PAS染色法用于检测小鼠肠道病理形态学改变,Lgr5、溶菌酶和Ki67免疫组化染色检测肠上皮增殖、分化、抑菌和再生能力的变化,同时,应用TUNEL凋亡荧光染色检测小鼠肠上皮凋亡水平的改变。为了了解肠道炎症水平的改变,采用Western blot法和ELISA法分别检测了肠道炎症相关蛋白HMGB1、TLR4、MyD88和NF-κB的表达水平和血清炎症因子IL-6、IL-8、TNF-α、INF-γ和 IL-10 的含量。为了研究肠道菌群及其代谢产物的变化对肠损伤的影响,采用高通量测序法分析了肠道菌群组成和功能,高效液相色谱法测定结肠内容物中短链脂肪酸(short chain fatty acids,SCFAs)的含量。结果:(1)体外实验:血根碱在0.1μM浓度范围内未观察到明显毒性;血根碱预处理可以减轻辐射对肠上皮IEC-6细胞增殖能力的影响;与照射组相比,血根碱预处理组维持了较好的克隆形成能力;此外,血根碱预处理抑制了辐射诱导的IEC-6细胞凋亡相关蛋白NF-κB、Caspase-8和Caspase-3的表达水平,流式细胞检测的凋亡实验也验证了这一点,发现血根碱预处理后凋亡细胞数较照射组有明显减少。(2)体内实验:与照射组比,血根碱预处理组显著改善了照射后小鼠的髓外造血功能和骨髓嗜多染红细胞微核发生率;血根碱预处理通过促进了照射后肠组织的修复和减少肠细胞凋亡,进而改善了RⅢ;病理及免疫组化结果显示,血根碱预处理恢复了肠绒毛高度和肠隐窝数目、减少了 TUNEL阳性细胞数目、增加了 Lgr5阳性细胞、溶菌酶阳性细胞和Ki67阳性细胞的数目;降低了照射后血清促炎因子IL-6、IL-8、TNF-α和INF-y的表达水平;进一步的机制研究显示,血根碱处理抑制了炎症信号通路HMGB1/TLR4/MyD88/NF-κB的激活。此外,血根碱预处理可维持受照小鼠肠道菌群的多样性和相对丰度,调节肠道菌群的组成、功能以及SCFAs的含量,继而减轻了RⅢ。结论:本研究证实了血根碱对RⅢ有明显的防护作用,其作用机制可能是:1)血根碱可抑制辐射诱导的炎症和细胞凋亡;2)血根碱可通过调控HMGB1/TLR4/MyD88/NF-KB信号通路改善RⅢ;3)血根碱可重塑辐射诱导的肠道菌群及代谢产物的紊乱,从而改善RⅢ。综上所述,本项研究从临床盆腹部恶性肿瘤放疗及核事故所致的放射性肠损伤这一棘手问题入手,从辐射致肠生物学功能障碍、继而引发的炎症反应、氧化损伤、细胞凋亡、肠道菌群及其代谢产物紊乱及其相关的分子调控机理这一系列研究角度出发,以放射性肠损伤的动物模型和细胞模型为实验对象,解析了小分子药血根碱在放射性肠损伤中的疗效,揭示了血根碱分别通过调控炎症HMGB1/TLR4/MyD88/NF-κB相关通路及重塑肠道菌群稳态两方面的功能作用,为临床放射性肠损伤防治提供了新思路。更多还原

【Abstract】 Objective:Radiation-induced intestinal injury(RⅢ)is one of the major side effects caused by radiation accidents or radiotherapy in patients with pelvic,peritoneal and retroperitoneal tumors.The primary pathogenesis of RⅢ includes injury to mechanical barrier,chemical barrier,immune barrier and biological barrier.Radiation gradually damages the structure of the small intestine and inhibits the function of absorption and protection.Lamina propria is exposed to the gut microbiota that induce acute inflammatory responses.This phenomenon causes nausea,vomiting,diarrhea,bleeding,and intestinal perforation,leading to death caused by bacterial infection and septic shock.RⅢseriously affects the patients’ quality of life and the curative effect of clinical tumors,and has limited effective therapies.Recently,the relationship between gut microbiota and diseases has been attracted by researchers.The radiation-induced dysbiosis of the intestinal microenvironment promotes susceptibility to intestinal injury.The regulation of the gut microbiota and its metabolites may be a feasible measure to reshape intestinal homeostasis and alleviate RⅢ.Therefore,protectors against RⅢ that can regulate the gut microbiota have received great attention in the field of radiation medicine.Sanguinarine(SAN),a natural small molecular compound,possesses numerous biological activities,such as antibacterial,anti-inflammatory,and antitumor.It is widely used in aquaculture as a drug feed additive.Our previous research demonstrated that SAN protects animals against radiation-induced acute damage to organs,especially in small intestine.However,the mechanism is not clear.In this study,we investigated the beneficial effects and underlying mechanisms of SAN on intestinal injury induced by ionizing radiation both in vitro and in vivo.Methods:(1)In vitro experiment:To explore the protective mechanism of SAN on radiation-induced injury at the cellular level,cell counting kit-8(CCK-8)was used to measure the cell viability of small intestinal epithelial cell line-6(IEC-6)at different concentrations of SAN.SAN was pretreated for 30 min before X-ray irradiation and the changes of IEC-6 cell viability and clonogenic ability under different treatments were analyzed.In addition,the expression levels of apoptosis-related protein NF-κB,Caspase-8,Caspase-3 and the relative percentage of apoptotic cells were detected via Western blot analysis and flow cytometry,respectively.(2)In vivo experiment:C57BL/6J mice aged 6-8 weeks were randomly divided into control group(CON),whole abdominal irradiation group(WAI)and SAN+WAI group.Mice were exposed to 10 Gy WAI to establish the RIII model.2.5 mg/kg SAN or equal volume normal saline was injected intraperitoneally 3 days and approximately 30 min before WAI.To explore the damage of hematopoietic system,histological examination was performed to assess the spleen injury,bone marrow micronucleus assay was used to analyze chromosome damage of bone marrow cells and the changes of peripheral blood cells were analyzed by blood routine test.To clarify the damage of intestinal system,histological examination was performed to assess the intestinal injury.Lgr5,lysozyme and Ki67 immunohistochemical staining were used to detect the changes of intestinal epithelial proliferation,differentiation,bacteriostasis and regeneration.TUNEL assay was used to detect the changes of intestinal epithelial apoptosis in mice.To investigate the changes of intestinal inflammation level,inflammation-related pathways and serum inflammatory cytokines were detected by Western blot and enzyme-linked immunosorbent assay(ELISA),respectively.To study the changes of gut microbiota and its metabolites,high-throughput sequencing was used to characterize the gut microbiota profile.High-performance liquid chromatography was performed to assess short-chain fatty acids(SCFAs)in colonic contents.Results:(1)In vitro,no obvious toxicity was observed less than 0.1 μM of SAN.SAN pretreatment reduced the effect of proliferation of IEC-6 cell induced by radiation.Compared with the irradiation group,SAN pretreatment group maintained normal clonogenic ability.Furthermore,SAN pretreatment inhibited the expression levels of apoptosis-related protein NF-κB,Caspase-8 and Caspase-3 in IEC-6 cells,which was verified by flow cytometry.The apoptotic cells after SAN pretreatment were less than those in the irradiation group.(2)In vivo,SAN pretreatment protected hematopoietic system by improving the extramedullary hematopoiesis(EMH)and the micronucleus incidence in the bone marrow in irradiated mice.SAN pretreatment alleviated RⅢ by promoting the intestinal recovery and reducing intestinal cell apoptosis.SAN pretreatment restored the height of intestinal villi and the number of crypts,reduced the number of TUNEL+cells,as well as increased the number of Lgr5+cells,lysozyme+ cells and Ki67+cells.SAN pretreatment reduced the levels of inflammatory cytokines such as interleukin-6(IL-6),interleukin-8(IL-8),tumor necrosis factor-α(TNF-α)and interferon-γ(INF-γ),and suppressed high mobility group box 1(HMGB1)/toll-like receptor 4(TLR4)/myeloid differentiation primary response 88(MyD88)/NF-κB pathway activation after WAI.In addition,SAN pretreatment reduced RⅢ by maintaining the diversity and relative abundance of gut microbiota in irradiated mice,regulating the composition and function of gut microbiota and the content of short chain fatty acids(SCFAs).Conclusions:Our findings demonstrated that SAN has obvious protective effect on RⅢ.The mechanisms include:1)SAN inhibits radiation-induced inflammation and against apoptosis;2)SAN improves radiation enteritis by down-regulating HMGB1/TLR4/MyD88/NF-κB pathway;3)SAN ameliorates RⅢ by remodelling the disturbance of gut microbiota and its metabolites induced by radiation.Thus,SAN is a potential therapeutic option for protecting against RIII in preclinical settings.更多还原