腹盆腔放疗诱发肠道微生态失调与肠源性感染的实验研究

Pelvic radiotherapy induces dysbiosis of gut microbiota and enteric infection in mice

目的 探索腹盆腔放疗照射对肠道微生态的影响及其与肠源性感染的关系.方法 模拟腹盆腔放疗照射BALB/c小鼠,2.0 Gy/d,连续照射5 d/周,分别于照射3周、5周和6周后停照1周的时间点收集回肠组织及其内容物样本.用实时定量RT-PCR检测抗菌肽和促炎性因子的表达;用PCR检测细菌在小鼠体内的移位情况;用变性梯度凝胶电泳技术检测分析肠道微生态的特征.结果 腹盆腔照射诱发了肠道潘氏细胞隐窝素-1和4表达紊乱,照射3周或照射6周后停照1周,小鼠回肠隐窝素-1和-4均呈现显著性降低（t=-7.43、-3.54、-4.72、-4.27,P＜0.05）;而照射5周小鼠回肠隐窝素-1和-4表达明显升高（t=6.15、5.75,P＜0.05）.放疗模拟照射3和5周时小鼠肠道微生物区系多样性指数和丰富度显著降低（t=-3.49、-4.19、-3.44、-4.97,P＜0.05）,呈现以乳酸杆菌等益生菌减少,大肠杆菌和弗氏志贺氏菌等条件致病菌增多为特征的微生态失调.受照小鼠肠系膜淋巴结和血液中的细菌DNA阳性率明显增高.照射3和5周后回肠组织IL-1β、IL-6和TNF-α显著性高表达（t=4.85、6.16、7.71、4.60、4.86、5.97,P＜0.05）;照射6周后停照1周时,肠道促炎性因子的表达量有所回落,但IL-1β和TNF-α的表达量仍显著性高表达（t=3.67、5.88,P＜0.05）.结论 腹盆腔放疗可诱发肠道抗菌肽表达紊乱,引起肠道微生态失调,进而导致肠源性细菌移位及感染性炎症的发生.微生态可能成为减轻放疗患者消化道不良反应的有效干预靶点.

Objective To explore the changes of gut microbiota in response to abdominal and pelvic radiotherapy and its potential relationship with intestinal infection.Methods Irradiation was delivered to the abdominal region of BALB/c mice,following the regular human pelvic-radiotherapy protocol,2.0 Gy/d,continuous 5 d/week.Samples of ileum tissue and the intestinal content were collected at different time points of irradiation procedure,including after 3 and 5 weeks,and at 1 week after 6 weeks of irradiation.Quantitative RT-PCR was used to measure the mRNA level of antimicrobial peptides and pro-inflammtory factors.Bacterial translocation was determined by PCR.The gut microbiota was characterized by the denaturing gradient electrophoresis assay.Results The expressions of cryptdin-1 and cryptdin-4 were decreased after 3 weeks of irradiation and at 1 week after 6 weeks of irradiation（t =-7.43,-3.54,-4.72,-4.27,P 〈 0.05）,while they were significantly increased at the 5 weeks of radiation （t =6.15,5.75,P 〈 0.05）.The diversity index and richness of gut microbiota after 3 or 5 weeks irradiation were significantly decreased （t =-3.49,-4.19,-3.44,-4.97,P 〈 0.05）.The gut microbiota dysbiosis of the irradiated mice was characterized with the decrease of probiotics of Lactobacillus and the increasing of opportunistic pathogen of Escherichia coli,Shigella flexneri,et al.Bacterial translocation episodes were more frequently in the irradiated mice than that of control animal.The mRNA levels of IL-1β、IL-6 and TNF-α were significantly increased after 3 or 5 weeks of irradiation （t =4.85,6.16,7.71,4.60,4.86,5.97,P 〈 0.05）.Compared with the control,the expression levels of IL-1β and TNF-α at the 1 week after 6 weeks of irradiation ending was also obviously enhanced （t =3.67,5.88,P 〈0.05）.Conclusions Pelvic radiotherapy can induce abnormality of enteric antimicrobial peptides and may result in gut microbiota dysbiosis.The disturbed gut microbial flora may further trigger an incurrence of bacterial translocation and enteritis.Therefore,the gut microbiota may be a potential interfering target to alleviate radiotherapy adverse effect.