Effects Of VDR On Ionizing Radiation-induced Intestinal Injury And Preliminary Molecular Mechanism

Background Radiation-induced intestinal injury is the main cause of early death of the injured in nuclear and radiation emergencies;it is also a common complication after radiotherapy for patients with tumors in the pelvis,abdomen or retroperitoneum.At present,there is no ideal treatment at home and abroad.Once it occurs,the mortality rate is almost 100%.Therefore,it is of great significance to study the prevention and treatment of radiation-induced intestinal injury,whether in the defense military or clinical tumor treatment.The mechanism of radiation-induced intestinal damage is complex,involving cell death,stem cell damage,immune activation,neuromodulation disorders,microcirculation disorders,etc.Therefore,its clinical treatment is very difficult,and there is no specific treatment method.Based on the complex mechanism of radiation-induced intestinal injury,whether to find drugs or targets that can intervene in the above multiple links has become the key to the treatment of radiation-induced intestinal injury.Vitamin D Receptor(VDR)is a nucleophilic protein that mediates the biological effects of vitamin D.In the past,the main role of VDR was to regulate calcium and phosphorus metabolism and promote bone development and maturation.However,in recent years,more and more studies show that VDR,as one of the highly expressed proteins in the intestine,plays a very important role in intestinal function maintenance,damage repair,inflammation regulation,stem cell differentiation regulation,and apoptosis inhibition.Therefore,we speculate that VDR may be of great significance in the prevention and treatment of radiation-induced intestinal injury,and this study has not been reported at home and abroad.Therefore,this article studied the role of VDR in radiation-induced intestinal damage.First,the relationship between VDR and intestinal radiation damage was clarified at the animal level,and then it was verified to intestinal epithelial cells and intestines by intervening VDR activity and regulating VDR expression Effects of Stem Cell Radiation Sensitivity.On this basis,the downstream molecular mechanism of action would also be studied in order to screen out the molecules that VDR regulates cell radiation sensitivity.Contents 1.To observe the effect of irradiation on the expression of VDR in the intestine of mice and the correlation between the degree of intestinal radiation damage and the amount of VDR expression.2.To study the effect of VDR intervention on radiation-induced intestinal injury in mice.3.To study the effects of VDR intervention and regulation of VDR expression on the radiation sensitivity of intestinal epithelial cells and intestinal stem cells.4.Screening and identifying the molecules of VDR regulating cell radiation sensitivity.Methods 1.Effects of ionizing radiation on the expression of VDR in intestinal tissues and cells of mice.1)Western blot and VDR immunohistochemistry were used to detect differences in the expression of VDR in the heart,liver,spleen,lung,kidney,and intestine of mice.2)Clarify the effect of ionizing radiation on the expression of VDR in intestinal tissues and cells.3)The mice were irradiated with 7Gy ?-rays,the VDR expression and the degree of intestinal radiation damage were quantified by immunohistochemistry,and the correlation between the two was analyzed by rank correlation test.2.Study on the effects of regulation of VDR on radiation-induced intestinal injury in mice.1)Enzyme-linked immunosorbnent assay(ELISA)was used to detect the effect of VDR agonist 24 h after administration on 1,25(OH)2D expression levels in mouse serum and intestinal tissue.2)Using Western blot experiments to clarify the agonistic effect of VDR agonists on the intestinal VDR of mice at 2h,4h,6h,10 h,24h and 48 h after administration.3)Count the 20-day survival period of mice after 7.5 Gy ?-ray irradiation after VDR agonist administration.4)H&E and fluorescein isothiocyanate(FITC)dextran experiment,Td T-mediated d UTP Nick-End Labeling(TUNEL)immunofluorescence,leucine-rich repeat-containing G-protein coupled receptor5(Lgr5+)fluorescence in situ hybridization and bromodeoxyuridine(Brdu)immunofluorescence clarify the effect of VDR agonist on radiation-induced intestinal damage in mice after 7Gy irradiation.5)Build a insufficient/sufficient vitamin D mouse model by diet feeding method,and use the ELISA method to detect the expression level of 1,25(OH)2D in mouse serum and intestinal tissue.Masson staining was used to observe the bone formation in mice.Tartrate Resistant Acid Phosphatase(Trap)staining was used to observe the number of osteoclast formation in mice.Western blot experiments were also used to verify the expression of intestinal VDR.6)Using H&E,FITC-dextran experiment,TUNEL immunohistochemistry,Brdu immunohistochemistry,Lgr5+ fluorescence in situ hybridization,Western blot to study the radiation-induced intestinal injury of model mice after 7Gy gamma irradiation.7)The intraperitoneal dose of VDR agonist is 2 ?g/?,and the relevant dose is irradiated 24 hours after administration.3.Effect of VDR on the radiation sensitivity of intestinal epithelial cells and small intestinal stem cells in vitro.1)Use the CCK8 experiment to determine the optimal drug dose for VDR agonists.2)Western blot experiments were used to determine the effect of VDR agonist on the expression of intestinal epithelial cells(Modek)VDR at 0h,0.5h,2h,4h,8h,and 24 h after administration.3)Clarify the effect of VDR agonist on the radiation sensitivity of Modek cells.Clone formation experiment: the irradiation dose of the irradiation group was 6Gy and 10 Gy,respectively;the dosage of the administration group was 100 n M.Detection of apoptosis: the irradiation dose of the irradiation group was 14Gy;the administration group was 100 n M;the apoptosis level of the cells was detected 48 h after irradiation.Cell cycle detection: the irradiation dose of the irradiation group was 8Gy;the administration group was 100 n M;the cell G2 M phase arrest was detected 8h after irradiation.5-ethynyl-2-deoxyuridine(Edu)flow cytometry/immunofluorescence detection of cell proliferation: the irradiation dose of the irradiation group is 14Gy;the administration group is 100 n M;the proliferation of cells was detected at 48 h after irradiation.4)Construct a VDR knockdown/overexpression cell line of Modek cells,and verify with real-time quantitative PCR(RT-q PCR)and Western blot experiments.5)The effect of knockdown/overexpression of VDR on the radiation sensitivity of Modek cells.Clone formation experiment: the irradiation dose of the irradiation group was 6Gy and 10 Gy,respectively.Detection of apoptosis: the irradiation dose of the irradiation group was 14Gy;the apoptosis level of the cells was detected 48 h after irradiation.Cell cycle detection: the irradiation dose of the irradiation group was 8Gy;the G2 M phase arrest of cells was detected at 8h after irradiation.Edu flow cytometry/immunofluorescence detection of cell proliferation: the irradiation dose of the irradiation group was 14Gy;the proliferation of cells was detected at 48 h after irradiation.6)Clarify the dose of VDR agonist and its effect on intestinal stem cells after irradiation.Small intestine organoid drug toxicity screening: After organoids are plated,the toxic effects of 10 n M,25 n M,50 n M,100 n M VDR agonists on organoids are detected.In vivo experiments: mice were given a single intraperitoneal injection of Calcitriol(VD)at a dose of 2 ?g/? in 24 h before irradiation.Subsequently,7 Gy of gamma rays were irradiated at a dose rate of 1 Gy/min.8h after irradiation,the intestinal organoids were extracted and plated for culture observation and immunofluorescence staining of Olfactomedin 4(OLFM4)and Edu.The mice in the control group were given the same volume of PBS and treated as before.In vitro experiment: The intestinal organs of mice were extracted and plated 24 h before irradiation,and then given 25 n M Calcitriol(VD)treatment.After 24 h,7Gy ?-ray irradiation was performed at a dose rate of 1Gy/min.Culture observation and immunofluorescence staining of OLFM4 and Edu were carried out.The control group was given the same volume of PBS and treated as before.Western blot was used to detect the potential molecular effects of ISC after VDR regulated radiation.Organoid culture was used to detect the sensitivity of vitamin D deficient mice to radiation.4.VDR regulates the radiation sensitivity of intestinal epithelial cells.1)The RNA of Modek-VDR-SH cell line was extracted by TRIZOL method,and differential gene screening was performed using RNA-SEQ.2)GO analysis and KEGG analysis were performed on the differential genes.RT-q PCR was used to verify some differential genes.3)si RNA interference related differential genes were used to observe the effects on Modek-VDR-OE cells after irradiation,so as to identify the downstream molecules of VDR.Results Effects of ionizing radiation on VDR expression in intestinal tissue and epithelial cells of mice.1.The intestinal VDR abundance is highest in all tissues and organs of mice.2.Ionizing radiation can induce VDR expression in intestinal tissues and epithelial cells of mice.3.Mice intestinal radiation damage is negatively correlated with VDR expression.Study on the effects of regulation of VDR on radiation-induced intestinal injury in mice.1.VDR agonists can alleviate radiation intestinal damage in mice,inhibit intestinal epithelial cell apoptosis,and prolong the survival time of mice after radiation.2.VDR agonists can protect intestinal stem cells and promote the repair of intestinal epithelium after irradiation.3.Vitamin D nutrient intake control build a deficient / high vitamin D mouse model.4.Intestinal radiation damage in mice deficient in vitamin D increases,while intestinal radiation damage in high-vitamin D mice decreases.5.The function of intestinal stem cells in mice lacking vitamin D is inhibited and damage repair is weakened;the repair of intestinal damage in mice with high vitamin D is significantly accelerated.Effect of VDR on the radiation sensitivity of intestinal epithelial cells and small intestinal stem cells in vitro.1.VDR agonists can up-regulate VDR expression in mouse intestinal epithelial cells.2.VDR agonists reduce radiation sensitivity of Modek and small intestinal stem cells.3.Knockout of VDR increases radiation sensitivity of Modek cells.4.Overexpressing VDR can reduce radiation damage in Modek cells.Preliminary study on the mechanism of VDR regulating the radiosensitivity of intestinal epithelial cells.1.Using Modek-VDR-SH as the research object,by sequencing the RNA-SEQ,we found that 232 genes were down-regulated and 158 genes were up-regulated after knocking down the VDR.GO analysis and KEGG analysis of differential genes were performed.2.Select some differential genes for verification.The RT-q PCR results are consistent with the sequencing results.3.Construction of differential gene si RNA,and found that interference with PDK1 and HIF2? can reverse the down-regulation of intestinal epithelial cell radiosensitivity induced by over-expression of VDR.Conclusion Ionizing radiation can induce the expression of VDR in the intestinal tissue of mice,while agonizing or up-regulating the expression of VDR can reduce the intestinal radiation damage in mice,reduce the radiation sensitivity of intestinal epithelial cells,protect the function of ISC,and promote the repair of IEC damage after irradiation.The protective effect of VDR on the intestine may be achieved by targeting the HIF / PDK1 pathway.