The Effects And Mechanisms Of Antibiotic Pretreatment On Acute And Chronic Radiation-Induced Intestinal Injury In Mice

Aims: The intestinal flora is involved in the damage of intestines and other organs under intestinal mucosa injury.Germ-free mice can significantly resist radiation-induced intestinal damage.In this study,we pretreated mice with metronidazole and quadruple antibiotics,then used 16S-r RNA sequencing to evaluate the effect of antibiotics on intestinal flora clearance,the changes of intestinal flora before and after radiation-induced intestinal injury and the effect of antibiotic pretreatment on reconstruction of the intestinal flora after radiation-induced intestinal injury.We further investigated the potential mechanism of antibiotic pretreatment on acute and chronic radiation-induced intestinal injury in mice.Materials and methods: In the first part,8-week-old male C57BL/6 mice underwent a single abdominal precision radiation at a dose of 10 Gy.The 16S-r RNA sequencing technology was used to analyze the intestinal flora and compare the changes of intestinal flora before and after abdominal radiation in mice.In the second part,mice were treated intragastrically with saline,metronidazole or quadruple antibiotics once a day for 14 days.After that,feces were taken for 16S-r RNA sequencing analysis to evaluate the effect of antibiotic treatment on intestinal flora and the efficiency of clearance.In the third part,16S-r RNA sequencing analysis was performed to analyze the intestinal flora of mice in each treatment group 3 months after abdominal radiation.The effect of antibiotic pretreatment on reconstruction of the intestinal flora after radiation-induced intestinal injury was evaluated.Based on the previous three parts,we used HE staining,Sirius red staining,?-SMA,ki67 and Tunel immunohistochemical staining to assess the effects of antibiotic pretreatment on acute and chronic radiation-induced intestinal injury in the fourth part.ELISA and immunoblotting was performed to explore its underlying mechanisms.Results: The first part revealed that Verrucomicrobia decreased from 2.9% to 0.0006% after radiation,and such changes significantly affected the diversity of the intestinal flora in mice.In addition,the alpha diversity and beta diversity of intestinal flora were significantly decreased after radiation,which was detrimental to the ecological balance of intestinal flora.Furthermore,radiation could lead to the overgrowth of some gram-negative pathogenic bacteria making them become the dominant flora in intestinal of mice,thus aggravating radiation-induced intestinal injury.The second part declared that quadruple antibiotics could effectively remove most of the intestinal flora in the feces of mice,imitating the intestinal environment of germ-free mice to some extent.On the other hand,the metronidazole treatment could only remove parts of the intestinal bacteria,aggravating the intestinal flora disorder and causing some pathogenic bacteria to overproduce,such as Escherichia coli and Shigella.In the third part,we found that quadruple antibiotics pretreatment would not weaken the reconstruction ability of intestinal flora in mice after radiation-induced intestinal injury.On the contrary,the diversity of intestinal flora in antibiotics-treated group after radiation was superior to that of the control group.The fourth part of the study indicated that quadruple antibiotics pretreatm ent could significantly improve the survival rate of mice after radiation,appar ently attenuated acute radiation-induced injury,and prevent intestinal fibrosis d uring chronic phase.Further study showed that these effects was achieved thr ough modulating the LPS/TLR4/My D88/NF-?B/macrophage polarization/TGF-?1/Smad-3 signaling pathways.Conclusions: Radiation-induced intestinal injury causes significant disturbances in the intestinal flora of mice.Quadruple antibiotics effectively remove intestinal flora and imitate the intestinal environment of germ-free mice,while the use of metronidazole only may cause intestinal flora disorder.Quadruple antibiotics pretreatment is beneficial to the reconstruction of intestinal flora in mice after radiation-induced intestinal injury.Quadruple antibiotics pretreatment improve the survival rate of mice after radiation,reduce radiation-induced intestinal injury and prevent intestinal fibrosis via LPS/TLR4/My D88/NF-?B/macrophage polarization/TGF-?1/Smad-3 signaling pathways.