Protective Effect And Mechanism Of Rac1 Inhibition On Radioactive Intestinal Injury

With the deepening of human understanding of ionizing radiation(IR),the use of which in national defense,commercial,agricultural,medical and other fields has become more and more extensive and common.,but we should clearly recognize the great harm of high dose IR to human body.Clinically,radiotherapy has become one of the combination therapy methods for most patients with malignant tumors,which can effectively alleviate and kill tumor cells,but the side effects caused by not only increase the economic pressure of the patients,more to reduce the patients' quality of life,which imposes a huge financial and physical burden on patients.Intestinal mucosa is very sensitive to IR because of its rapid self-renewal ability.Radiation-induced intestinal injury(RIII)is a common complication of radiotherapy in patients with pelvic,abdominal and retroperitoneal tumors,and is also the main cause of death of initial nuclear radiation in nuclear radiation events.It is generally believed that RIII is caused by both direct and indirect effects of IR.IR can directly damage the structural integrity of cell DNA and proteins and inhibit their synthesis.Meanwhile,the production of large amounts of reactive oxygen species(ROS)can also cause damage to cells and eventually lead to apoptosis.There is no specific drug for RIII in clinic,which can protect normal tissues and also have protective effect on tumors.Therefore,it is of great clinical significance to find a drug that can not only prevent normal tissue ionizing radiation damage,but also have no protective effect on tumors.Ras-related C3 botulinum toxin substrate 1(Rac1)is a small molecule Guanosine Triphosphate(GTP)binding protein,a classic member of the Rho family.Rac1 is associated with many cellular behaviors,including apoptosis,differentiation,and gene transcription etc.Studies have found that Rac1 is not only a key component of NADPH oxidase(NOX)activation,which is a key enzyme for the production of ROS in cells,but also an important molecule that affects the cell cycle.The large increase of ROS and cell cycle arrest are known to be important mechanisms for ionizing radiation damage and repair.Therefore,we speculated that Rac1 may play an important role in the regulation of intestinal ionizing radiation injury,which has not been reported at home and abroad.In this study,the relationship between Rac1 and intestinal ionizing radiation injury was studied.The role of Rac1 was verified through in vitro and in vivo experiments,and the mechanism was further discussed.In addition,we conducted experimental studies on Rac1 radiosensitivity of intestinal tumor cells,so as to more comprehensively evaluate the potential application value of Rac1 molecule as a therapeutic target for intestinal tumor radiotherapy injury.We first tested the effect of Rac1 inhibition on RIII in mice.H&E staining results showed that after irradiation,in the control group the number of intestinal crypts decreased,the intestinal mucosa decreased and the intestinal wall became crowded and expanded due to capillary dilatation and inflammatory cell infiltration,while the mice in the treatment group significantly improved(P<0.01),with less intestinal mucosa damage,more crypts,and mild inflammatory response.This suggests that inhibition of Rac1 activity before irradiation can protect the intestinal damage caused by radiation.Subsequently,apoptosis in the small intestine was detected,and the results of TUNEL staining and Western Blot both showed that the apoptosis in the reatment group was significantly lower than that in the control group(P<0.01).The results of animal experiments confirmed our hypothesis that Rac1 is associated with radioactive intestinal injury,and inhibition of its activity can protect intestinal tissues and reduce damage and apoptosis of intestinal structures.On the premise that inhibition of Rac1 activity at the animal level could protect the irradiated intestinal tissue,MODE-k,mouse intestinal epithelial cell line,was used to conduct a more comprehensive study and preliminarily explore its mechanism.Cell viability assay showed that inhibition of Rac1 activity increased MODE-K cell vitality(P<0.01),and an appropriate dose concentration of 100?M was determined for subsequent experiments.The results of flow cytometry and Western Blot showed that the apoptosis rate and expression of apoptotic proteins in the treatment group after irradiation were significantly lower than those in the control group(P<0.01).The results of mitochondrial membrane potential(MMP)experiment showed that the MMP of the drug group was significantly higher than that of the control group(P<0.01).These results suggest that inhibition of Rac1 activity can reduce the radiosensitivity of intestinal cells in normal mice,and inhibit the activation of mitochondrial apoptosis pathway after irradiation.In addition to protecting normal tissues,the radiation protection agents given to tumor patients during radiotherapy also have a protective effect on tumor tissues.A large number of previous literatures reported that Rac1 was either overexpressed or activity significantly increased in tumor patients,and some studies even considered Rac1 gene to be a proto-oncogene.Therefore,in addition to relevant experiments on normal animals and cells,we also tested the effect of Rac1 inhibition on mouse colon cancer cell line,CT-26.The results showed that the activity of tumor cells in the reatment group was significantly decreased(P<0.01),and the effect of inhibitor concentration of 100?M was the best,so we chose 100?M as the concentration of drug administration in subsequent experiments.The results of flow cytometry and Western Blot showed that the apoptosis rate(P<0.05)and apoptosis-related protein expression(P<0.01)were significantly higher in the treatment group than in the control group.The results of mitochondrial membrane potential experiment showed that the mitochondrial membrane potential in the treatment group was significantly higher than that in the control group(P<0.05).This indicated that inhibition of Rac1 activity could increase the radiosensitivity of tumor cells.The results were contrary to those of normal cells,indicating that inhibition of Rac1 had radiation protection effect on normal cells,but increased radiation sensitivity of tumor cells.After obtaining the above results,we carried out further studies on the molecular mechanism of inhibiting Rac1 to reduce the radiation damage of intestinal epithelial cells,and also explored the reasons for the different effects of inhibiting Rac1 between normal cells and tumor cells.We found that inhibition of Rac1 activity reduced ROS levels in intestinal epithelial cells and changed the expression of Cylin D1 and Cyclin B1 after irradiation,prolonged the G1 phase to repair damaged DNA,thus shorten the G2/M phase and thus reducing the cell cycle arrest after irradiation.However,for intestinal tumor cells,we found that inhibition of Rac1 activity could not effectively reduce ROS levels in tumor cells after irradiation,but significantly prolonged the G2/M phase arrest and skipped vital G1 phase DNA repair,leading to longer cell cycle and increased apoptosis.These results indicated that Rac1 regulation of cell cycle and ROS showed significant differences between normal cells and tumor cells,which was probably caused by Rac1 mutation in tumor cells.To verify this hypothesis,we also sequenced the Rac1 genes in normal intestinal epithelial cells and intestinal tumor cells,and found that Rac1 mutations did exist in tumor cells.In summary,we found that inhibition of Rac1 can effectively protect intestinal tissues and epithelial cells and reduce their sensitivity to ionizing radiation.At the same time,Rac1 inhibition has no protective effect on tumor cells and increases the radiosensitivity of tumor cells.The above findings are of great significance for radiotherapy of clinical tumor patients and have important reference value for the prevention and treatment of radiation injury in nuclear accidents.