Alteration Of Intestinal Microbiota And Enteritisgenesis In Mimic Abdominopelvic-radiated Mice

Nowadays, increasing number of patients suffer from cancer owing to ever serious environment pollution. Surgery, chemicaltherapy and radiotherapy are three main cancer-therapy adopted in world-wide hospitals. With the wide utilization of radiation in medicine field, more and more cancer patients received radiotherapy. Hower about 40% patients received abodominopelvic-radiation therapy suffered from side-by effect of ionization. Small intestine is one of the organs sensitive to radiation. Either total body/abdomen exposure to a certain dose of radiation can result in the intestinal dysfunction, thus developing into the radioation enteritis, which often seriously affect the living quality, physiological and psychological status of patients. However, the detailed pathogenesis of radiation enteritis is yet to be elucided.There are over 103 kinds of microbes colonizing in the human gut (1014 CFU), constituting the complexed microbiome. Under physical conditions, the homostasis of mutual interaction between gut microbiota and intestinal mucosal immunity is keeped balance. While, as the body was disturbed by some stimulus, the gut homeostasis could be perturbed. Then, intestinal mucosal could be invade by opportunistical pathogens residing in the digestive tract. Previously, several studies have demonstrated that the epithelial cells would be partially detachmented when the whole body was exposed to a certain dosage of ionizing radiation (>10 Gy), which led to the occurance of necrosis and the damage of mucosal barrier. Meanwhile, the component and structure of gut microbiota may be changed. Opportunistic pathogens would then penetrate gut mucosal barrier and release a large amount of toxins. This is associated with the radioactive enteritis, endotoxemia, and multiple organ dysfunction syndrome. Therefore, the alteration and regulation of gut flora is closely related to the occurrence and development of radioactive enteritis.In clinical, about 20% cancer patients received abodominopelvic radiation-therapy developed a series of radioactive symptoms, such as decreased appetize, vomit, abodomen pain and/or diarrhea et al. We hypothesize that these symdrome would be associated with gut microdrome dysbiosis instigated by radiotherapy ionization. Human -defensin 5 is an endogenous defense peptide secreted by Paneth cells locating at the base of intestinal villi. It plays a crucial role in maintaining the homeostasis of gut flora, and suppresses the infection of opportunistic pathogens. Previously study demonstrated that the patients whose HD5-encoding gene was mutated or reduced expression were susceptible to the infective enteritis. Moreover, experiments in mice showed that the expression of Cryptdin 4 (Crp4), which was expressed in mice and homologous to HD5, was significantly changed in inflammatory bowel disease. Neverthless, when intestine is exposed to clinical radiotherapy radiation (2 Gy/day,5 day/week for 5-6 weeks), the alteration of antimicrobial peptide expression still remains unkown. Moreover, what changes and how to alter of human intestional microbiota during the radiotherapy procedure is yet to explore. Additionally, the underlying mechanism and the potential relevance with radioactive enteritis also require further study.In this study, using the mimic abodminopelvic radiotherapy mouse model, we firstly evaluated the component and structure of intestinal microbiota by bacterial 16s RNA sequencing technology. Meanwhile we will measure the status of proinflammatory cytokines in intestinal tissue and serum. And then the association between changed gut microbiota and inflammatory respone in mice will be analyzed. The aim of this study is to explore the mechanism of intestinal microdrome dysbiosis and radioactive enteritisgenesis. The main results and conclusions obtained are as follows:1. The protocol of clinical radiotherapy for cancer patients was adopted, and mimic abdominopelvic radiation mice model were successfully established. The BALB/c mice were confirmed as appropriate model for gut microbiota and radioactive enteritis.2. The result from bacterial 16s RNA macro-genome sequencing showed the richness and diversity of samples from both small intestine and large intestine fragments were significantly decreased during radiotherapy period. The gut microbiota dysbiosis was confirmly induced by clinical radiation dose.3. The analysis of macro-genome from gut flora displayed that the richness of firmicutes were significantly decreased, and Bacteroidetes was dramatically increased, namely the ratio of Firmicute to Bacteroidetes changed reciprocally.4. The analysis of genome showed that the gut microbiota at family and genus levels happened significantly changes during radiotherapy peroid. The results showed that some opportunistic pathogens were increased. These opportunistic pathogens harbored Escherichia-Shigella, Enterobacteriales, Staphylococcus, Streptococcus et al. while, some probiotics, such as Lactobacillus, were decreased. The value of colonizing resistance of gut flora was obviously decreased.5. It was found that the positive rate of bacterial DNA in intestinal mesentery nodes and blood from radiation mice were significantly increased. This suggested that radiotherapy increased the occurrence of intestine-pathogen infection.6. The results of quantitative RT-PCR showed that proinflammatory cytokines of IL-1β, IL-6 and TNF-a in ileum of radiation mice were significantly increased. Protein chip testing found that the levels of TNF-a, IL-1β, MCP-1, KC and LIX increased with increasing radiation dose. The resemble tendancy of serum LPS was also observed. These results showed that intestinal and systemic inflammatory responses were induced by radiotherapy.7. The close correlations between the relative richness of gut Escherichia and serum LPS level, TNF-a, IL-1β were observed. This analysis further confirmed that inflammatory response of radiated mice was associated with the gut microbiota dysbiosis characterized with higher richness of Escherichia.8. The results of quantitative RT-PCR and immunohistochemical observation displayed that the expression of gut defensins presented fluctuating in the period of radiotherapy. This suggested that the gut microbiota dysbiosis would attributed to the chaos of intestinal antimicrobial peptides.9. The increased apoptosis rate of intestinal epithelial cells and enhanced occurrence of autophagy in Paneth cells were also observed in mice received mimic abodeminopelvic radiotheary.