| Background Radiation Enteritis(RE)is an intestinal injury disease caused by Radiation(X-ray,gamma ray,etc.)for patients with pelvic,abdominal and retroperitoneal malignant tumors,which can affect the small intestine,colon and rectum.It can cause abdominal pain,diarrhea,constipation,intestinal obstruction,intestinal fistula,and even death of the patient,seriously affecting the quality of life of the patient.The incidence of the disease is high at home and abroad,and it is increasing year by year.One of the main factors of radiation damage is the excessive production of oxygen free radicals,a large amount of oxygen free radicals can cause the occurrence of cell function damage and trigger a cascade reaction leading to cell apoptosis.Nrf2/ARE signaling pathway is an important endogenous anti-oxidative stress pathway in the body,which is closely related to the occurrence and development of a variety of diseases.And this signaling pathway has been proved to protect tissues from oxygen free radical damage in liver,lung,kidney and other tissues,but there few reports on radiation intestinal injury.At present,most of the studies on radiation-induced intestinal injury by X-ray are from clinical case observation,and the mechanism of injury is still unclear.There are few experimental studies on the pathogenesis,and the main reason for the lack of experimental research is the lack of suitable experimental model.Therefore,the exploration of experimental modeling conditions for radioactive intestinal injury is the primary research content of this topic.Secondly,whether Nrf2/ARE signaling pathway has a protective effect in intestinal tissues and its mechanism is the focus of this topic.objective 1.By exploring the optimal conditions for RE model establishment,animal and cell models of acute radiation intestinal injury conforming to clinical and pathological characteristics were established,laying a foundation for the exploration of mechanism of radiation intestinal injury and the research and development of effective therapeutic drugs.2.By studying the dynamic expression and distribution of Nrf2/ARE signaling pathway in radioactive intestinal injury,the relationship and role of this signaling pathway with radioactive intestinal injury were clarified.3.Through selective intervention to regulate gene expression of Nrf2/ARE signaling pathway,the protective effect of Nrf2/ARE signaling pathway in radioactive intestinal injury and its molecular mechanism were preliminarily explored,providing new targets and ideas for clinical treatment of radioactive intestinal injury.Methods 1.Male SD rats and IEC-6 cell lines were irradiated with 6MV-X ray at different doses,(1)animal model: in the aspect of macrozoology,the mortality,changes of food intake and water intake,weight change and diarrhea of rats were observed,and intestinal mucosal tissue damage was observed by H&E.Changes in oxidative stress indexes(SOD activity,MDA content,GSH-Px activity)of jejunum tissues were detected with the kit.Immunohistochemistry and western blotting were used to detect the expression of apoptotic protein NF-κB in jejunum.(2)cell model: CCK-8 method was used to detect cell viability,and the optimal radiation dose and observation time point were finally screened out,and the animal and cell model conditions of radiation intestinal injury were established.2.(1)Dynamic expression and distribution of Nrf2/ARE signaling pathway in radioactive jejunum tissues: Male SD rats were randomly divided into a normal control group and a 10 Gy radiation dose group.Rats were sacrificed at 1h,6h,12 h,24h,48 h,60h,72 h,84h,96 h,120h,144 h and 168 h before and after irradiation,respectively.The intestinal injury and H&E staining of rats at different time points were observed to analyze the dynamic changes of intestinal mucosa.The dynamic expression of Nrf2 and its downstream molecule HO-1 m RNA and protein in intestinal tissues were detected by q PCR and Western Blot.The expression of Nrf2 and HO-1protein was detected by immunohistochemistry 48 h after irradiation.(2)The effect and significance of Nrf2/ARE: Nrf2 inhibitors inhibited the expression of Nrf2 m RNA in IEC-6 cells,and CCK-8 method was used to detect the cell viability,cell apoptosis was detected by Hoechst33258,and oxidative stress indexes such as intracellular ROS change,SOD activity,MDA content and GSH-Px activity were detected by the kit,so as to further clarify the role and significance of Nrf2 in radiation intestinal injury.(3)whole transcriptome gene sequencing was used to analyze the whole gene changes and Nrf2-related gene differences in jejunum tissues of normal rats and 10 Gy irradiated rats.3.To explore the protective effect of Nrf2/ARE signaling pathway in radiation intestinal injury and its molecular mechanism: P38 MAPK inhibitor,ERK inhibitor and PI3 K inhibitor were used to intervene IEC-6 cells respectively,and the cell activity,oxygen free radical,cell apoptosis,Nrf2 protein and m RNA expression in normal cells and irradiated cells of different intervention groups were detected.Results 1.The establishment of the acute radioactive intestinal injury animal model: the results showed that the exposure dose for under 6Gy,change the indicators such as zoology,oxidative stress,histopathology of rats,there was no significant difference compared with normal rats,the dose over 6Gy,with the increase of irradiation dose,significantly increase the rat intestinal injury,dose of 12 Gy,rat survival rate is only 20%,with the passage of observation time after irradiation,72 h after irradiation damage of rats is one of the most serious,recover gradually after 84 h.After comprehensive consideration of various indicators,10 Gy radiation dose can replicate animal models,which is similar to clinical RE symptom.According to the intestinal mucosa histopathology,oxidative stress and NF-κB and other indicators,it can be concluded that the injury is most serious at 72 h after irradiation.The intestinal mucosal histopathology,oxidative stress and NF-Κb and other indicators were the most severe at 72 h after irradiation.Therefore,the conditions for the establishment of an animal model of acute radiation intestinal injury were as follows: 6MV-X ray medical linear accelerator,400mu/min dose rate,10 Gy single irradiation,100 cm from the source skin,72 h after irradiation.(2)establishment of cell model of acute radiation intestinal injury: the results showed that: CCK-8 cell viability experiment showed that there was no significant difference in cell morphology and cell viability between the 8Gy irradiated cells and the normal group.When 12 Gy irradiated cells,nuclear shrinkage and lysis death occurred in 80% of cells;However,cells exposed to 10 Gy radiation showed the most significant damage 48 h after irradiation,with morphological changes,cell death rate of 50% and cell viability reduction of 50%.Compared with normal cells,the cells showed both difference and 50% survival rate and viability.Therefore,the conditions for the establishment of the cell model of acute radiation intestinal injury were: 6MV-X ray medical linear accelerator,400mu/min dose rate,10 Gy radiation dose single irradiation,the distance from the cell to the radiation source was 100 cm,and the observation time point was 24 h after irradiation.2.(1)The dynamic changes and expression of Nrf2 in RE of rats were studied.The results showed that the intestinal injury of rats was aggravated with the extension of irradiation time.At 72 h after irradiation,intestinal injury was the most significant,with intestinal edema and congestion,intestinal mucosa swelling and shortened villi.Nrf2 and the HO-1 m RNA and protein expressions were significantly increased at 1h after irradiation,reaching peak at 48 h and 12 h after irradiation respectively,and decreased at 72 h and 24 h after irradiation,but there were still significant differences compared with the normal group.(2)The protective effect of Nrf2 on RE in rats.The results showed that after the intervention of Nrf2 inhibitor,compared with the single irradiation group,the damage of intestinal epithelial cells was significantly aggravated,cell activity was decreased,ROS content and apoptosis were significantly increased,and oxidative stress reaction was significantly increased.(3)whole transcriptome gene sequencing analysis experiment,the results showed that: the model group of rats have 5284 genes changed,more than 300 genes have significant changes,comprehensive analysis and Nrf2-related genes,including HO-1,MAPK,ERK and PI3 K pathway have significant changes.3.Explore Nrf2 / ARE signaling pathways in the protective effect and its molecular mechanism in the RE,the results show that different inhibitors after intervention Nrf2 upstream approach,compared with irradiation group(IR),IEC-6 radiation damage cells,P58 MAPK inhibitors and ERK inhibitors group cell vitality,oxygen free radical,apoptosis,Nrf2 protein and m RNA expression changes were not statistically significant,and the PI3 K inhibitor intervention group had significant damage,cell viability decreased significantly,oxygen free radicals and cell increased significantly,Nrf2 m RNA and nucleoprotein expression were significantly reduced.Conclusions 1.(1)The conditions of the animal model simulating clinical radiation intestinal injury were: 6MV-X ray medical linear accelerator,400mu/min dose rate,10 Gy radiation dose single irradiation,source skin distance of 100 cm,observation time point of 72 h after irradiation;(2)the conditions of the cell model of radioactive intestinal injury were: 6MV-X ray medical linear accelerator,400mu/min dose rate,10 Gy radiation dose single irradiation,the distance from the cell to the radiation source was 100 cm,the observation time point was 48 h after irradiation.2.(1)Nrf2/ARE signaling pathway plays an important protective role in radiation intestinal injury,which is activated in the early stage of injury(1h after irradiation)and the highest expression level at 48 h after irradiation.Its downstream antioxidant and detoxifying enzymes HO-1 were up-regulated.(2)Nrf2 is a self-protective mechanism of radiation intestinal injury.After inhibiting Nrf2 pathway,radiation intestinal injury significantly increases.3.PI3K/Akt pathway plays a major role in the activation of Nrf2/OH-1 pathway in radiation intestinal injury. |
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