| Background: Radiation enteritis(RE)is one of the most serious side effects of the digestive system during radiotherapy for malignant tumors such as pelvic and abdominal cavities,which can lead to a decrease in patients’ tolerance to radiation therapy and quality of life.With the wide application of radiotherapy technology in malignant tumors,its side effects on the paracancerous tissues are getting more and more attention.The intestinal epithelial cells are sensitive to radiation due to rapid proliferation,and some patients receiving radiation therapy will develop radiation enteritis.So far,the clinical treatment of radiation enteritis with drugs such as antibiotics and anti-inflammatory agents is not effective,and the related basic research and drug development are not ideal for effective prevention and treatment,and the incidence of radiation enteritis is increasing with the increase in the number of patients treated with radiation for malignant tumors.The establishment of acute radiation enteritis models is based on animal experiments,mainly in rats,dogs and rabbits,but due to the influence of the selection of experimental animals,modeling apparatus,positional methods,different irradiation sites and irradiation doses,and the poor reproducibility of experimental results,the establishment of a suitable radiation enteritis model has become the key to research.In this study,we established radioenteritis models at different doses and time points,and explored the types of irradiation-induced death of normal intestinal epithelial cells and the molecular mechanisms at the cellular level,aiming to provide some molecular mechanisms for the development of radioenteritis.Part I.Establishment of a mouse model of acute radiation enteritis and study of intestinal epithelial cell death and inflammatory factor changesObjective: To irradiate C57BL/6 mice with different doses of medical linear gas pedal 6 MV-X rays to establish a mouse model of acute radiation enteritis and to study the changes of intestinal epithelial cell death and inflammatory factors.Methods: Twenty-three SPF-grade male C57BL/6 mice aged 6-8 weeks were randomly divided into control(n=3)and 6,12,15 and 18 Gy(n=5)radiation groups at different doses to observe the survival rate and weight change of mice for 15 days.Another 60 mice were randomly divided into control group and 6 Gy,12 Gy,15 Gy(n=15)different dose radiation groups,and 5 mice were executed by cervical dislocation on day 1,3 and 7 respectively,and the terminal ileum tissues were taken for pathological sectioning,HE staining and Masson staining,to observe the pathological histological changes and establish the mouse model of acute radiation enteritis.RT-q PCR was used to detect the changes of m RNA relative expression of pro-inflammatory cytokines IL-1β,IL-6,TNF-α and pro-fibrotic growth factor TGF-β1 in intestinal tissues,and immunohistochemistry was used to detect the expression of cleaved-PARP in intestinal tissues to observe the changes of inflammatory factors and cell death in intestinal tissues after irradiation.Results: Compared with the control group,mice in each radiation group showed different degrees of health problems and weight loss after irradiation,especially in the 15 Gy group,but no mice died,and the mortality rate of mice in the 18 Gy group was 60% within 15 days.The corresponding phenotypes were not obvious in the 6 Gy and 12 Gy radiation groups.Therefore,the radiation conditions for acute radiation enteritis were established as follows: medical type linear gas pedal 6 MV-X-rays,source skin distance 100 cm,15 Gy one-time abdominal irradiation.On day 3 after irradiation,the 6 Gy,12 Gy,and 15 Gy radiation groups showed significant changes in pro-inflammatory cytokines IL-1β,IL-6,TNF-α,and pro-fibrotic growth factor TGF-β1 compared with the control group,and immunohistochemical results showed positive expression of cleaved-PARP in the intestinal tissue of the 15 Gy radiation group.Conclusion: 6 MV-X-ray one-time abdominal irradiation,gross observation and pathological sections showed that the 15 Gy and 18 Gy radiation groups had symptoms of clinical acute radiation enteritis,and the mice in the 18 Gy radiation group showed mortality,which could not provide a model for further experimental studies,and 15 Gy could be the best irradiation dose for the mouse model of acute radiation enteritis.With the increase of irradiation dose,the intestinal crypt cell damage was gradually severe,and the m RNA expression level of inflammatory factors also increased,and the intestinal tissue immunohistochemistry cleaved-PARP expression was positive.Part II.Preliminary study of the molecular mechanism of radiation-induced intestinal epithelial cell death at the cellular levelObjective: To preliminarily investigate the molecular mechanism of radiation-induced intestinal epithelial cell death at the cellular level.Methods: IEC-6 cell line was selected,and the cells were de-irradiated with medical linear gas pedal 6 MV-X rays at different doses of 5 Gy,8 Gy,10 Gy,and 12 Gy.Two methods,CCK8 method and PI single staining,were used to determine the optimal irradiation dose and the optimal observation time point,based on which reactive oxygen species ROS and flow Annexin V/PI double staining experiments were used to preliminarily determine the radiation-induced death of intestinal epithelial cells and the molecular mechanism of the mode of cell death induction.Results: At the cellular level,CCK8 experiments showed that the cell viability of IEC-6 cells decreased to about 50-60% at 72 h under 12 Gy irradiation conditions.PI singlestaining experiments showed that the cell death rate of IEC-6 cells was around 40% at 24 h under 12 Gy irradiation conditions.Combining the two experimental methods,as well as the best cell survival rate of 60% as reported in the relevant literature,a 6MV-X-ray medical type linear gas pedal with a radiation source 100 cm away from the cells was selected for the modeling dose and time of IEC-6 cells under a single irradiation of 12 Gy at 24 h.Western Blot results showed that activation of caspase3 occurred after cell irradiation and The addition of caspase inhibitor Q-VD-OPh resulted in a significant decrease in ROS release and apoptosis,a significant increase in cell viability,and a decrease in activated caspase3 protein expression in the cells.Conclusion: Medical linear gas pedal 6 MV-X-rays irradiated cells in a single session,and the combined results of CCK8 experiments and PI single-staining experiments indicated that 12 Gy,24 h was the modeling dose and time for IEC-6 cells,and that irradiation induced apoptosis in intestinal epithelial cells,which was associated with caspase3 activation.Figure [16] Table [10] Reference [78]... |
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