Alteration And Significance Of Small Intestinal Cryptdin4 In Ionizing-radiated Mice

With the wide utilization of nuclear technology in the industry, agriculture, medicine and military areas, the radiation injury turns to be a common occurence. Small intestine is one of the organs sensitive to radiation in human body. Either total body or abdomen exposure to a certain dose of radiation can result in the intestinal dysfunction, thus developing into the radioactive enteritis and seriously affecting the living quality of patients. According to the clinical statistics, the incidence of radioactive enteritis in patients with abdominal neoplasms and received radiotherapy is more than 50%. However, the definite pathogenesis of radioactive enteritis is still far from clear.There are more than 1000 kinds of microbes located in the human gut(1014 CFU) in human body, constituting the intricate microbiome. The balance between conditioned pathogen and probiotics is closely related to the health. Previoulsy, several studies have demonstrated that the epithelial cells would be partially detachmented when the small intestine was exposed to ionizing radiation, which led to the occurance of necrosis and the damage of mucosal barrier. Meanwhile, the amount and proportion of intestinal flora may be changed. Conditioned pathogens would then infect the host through the mucosal barrier and release a large amount of toxins. They are associated with the radioactive enteritis, endotoxemia, and multiple organ dysfunction syndrome(MODS). Therefore, the alteration and regulation of intestinal flora are closely related to the occurrence and development of radioactive enteritis.Human ?–defensin 5(HD5) is an endogenous antimicrobial peptide secreted by Paneth cells which locates at the base of intestinal villi. It plays a crucial role in maintaining the homeostasis of intestinal flora and suppresses the infection of conditioned pathogens. It has been previously pointed out that the people whose HD5-encoding gene was site mutated or reduced in copy number were susceptible to the infective enteritis syndrome. Moreover, animal experiments 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 subjected to radiation injury, the alteration of Crp4 expression still remains unkown. Additionally, the underlying mechanism and the potential relevancy with radioactive enteritis also require deeper study.In the present study, using the acute radiation injury mouse model, we firstly assessed the changes of the expression of Crp4 in small intestinal, the composition of intestinal flora, and the concentration of lipopolysaccharide(LPS) in serum after ionizing radiation. Compared with the germ-free mice after radiation injury, we preliminarily revealed a possible mechanism for the changes of the expression of Crp4 after radiation injury in normal mice. Besides, we analyzed the antibacterial activities of Crp4 against different species of bacteria and its LPS-neutralization effect. The aim of this study is to explore the effect of Crp4 expression changes on radiation enteritis. The main results and conclusions obtained are as follows:1. BALB/c mice received 8 Gy X-ray irradiation was confirmed more appropriate for the establishment of the intestinal acute radiation injury model.2. As assayed byimmunohistochemistry and Western blot, the expression of Crp4 dramatically increased in the small intestine and Paneth cells after irradiation, and it reached to the peak level at the 3th day after irradiation.3. The result of 16 S RNA gene sequencing showd that the composition of intestinal flora significantly was changed after radiation injury. The proportion of bacteroidetes and firmicutes were significantly decreased, while the proportion of proteobacteria was dramatically increased, which is mainly responsible for the release of LPS.4. As determined by limulus chromogenic assay, the content of LPS in the serum of mice after radiation injury was significantly increased.5. It was found that the expression of Crp4 in normal mice after intraperitoneal injection of LPS was significantly increased, while there was no significant changes in germ-free mice.These data suggested that the alteration of the number and proportion of G- bacteria in small intestine after radiation injury might be responsible for the raised expression of Crp4.6. The antibacterial assay in vitro showed that Crp4 possessed a selective antibacterial activity against microbes, where the conditioned pathogens were more sensitive than the probiotics in intestine. It indicates that the increased expression of Crp4 after radiation injury may play an important role in regulating the composition of intestinal flora.7. Protein chip testing found that the content of proinflammatory cytokines, including IL-1α, IL-1β, IL-6 and TNF-α,were significantly increased in mice serum after radiation injury.8. It was confirmed that LPS is able to induce mouse peritoneal macrophage cells to secrete IL-1β and TNF-α, whereas Crp4 can significantly inhibit the ability of LPS, suggesting that the increased expression of Crp4 after radiation injury might be an instrumental response to prevent excessive intestinal inflammation.