Effects Of GSDMD On Ionizing Radiation Damage Of Hematopoietic System And Intestine And Preliminary Molecular Mechanism

BackgroundThe prevention and treatment of ionizing radiation damage is a vital technical problem that must be solved in the safe utilization of nuclear energy and also one of the most important research topics in the field of radiobiology.Acute radiation syndrome?ARS?includes hematopoietic type and intestinal type,two of which are the most important types of acute radiation sickness.Currently,there is no other effective prevention and treatment method except the WR-2721 approved by FDA.Therefore,it has become the focus and difficulty in the field of nuclear radiation research to find the radiation protective agents and the key molecules in the radiation damage.In the previous studies,we found that the ligand LPS of TLR4 had a strong radiation protection effect,but the toxicity limited its clinical application.In 2014,Shao Feng reported that the intracellular receptor Caspase-11 of LPS activated the pyrotosis pathway,which has been widely studied and reported subsequently,where GSDMD acting as the executive molecule is the key to the occurrence of pyrotosis.And there has been no reports in the literature about relationship between GSDMD and ionizing radiation.Therefore,we took GSDMD as the research object,explored the role of GSDMD in the hematopoiesis and intestinal ionizing radiation damage,clarified the relationship between GSDMD and ionizing radiation,and preliminarily studied the molecular mechanism of GSDMD participating in ionizing radiation damage.Purpose1.Determine the effects of GSDMD on the hematopoiesis ionizing radiation damage;2.Determine the effects of GSDMD on the intestinal ionizing radiation damage;3.Clarify the molecular mechanism of GSDMD in intestinal ionizing radiation damage preliminarily.Contents,methods and results1.Effects of GSDMD on the hematopoietic ionizing radiation damage1.1 GSDMD KO significantly improved the survival period of ionizing radiation and protect peripheral blood injury.The survival rate of GSDMD KO mice was significantly improved after 30 days of irradiation.Peripheral blood injury was less severe and recovery was faster.1.2 GSDMD KO had a protective effect on bone marrow/spleen and thymus.After ionizing radiation,the number of nucleated cells in bone marrow/spleen/thymus of GSDMD KO mice was significantly higher,the bone marrow damage was reduced,and the spleen coefficient was less changed.1.3 GSDMD KO increased LSK cells and promoted the differentiation of myeloid progenitor.In GSDMD KO mice,the number and proportion of LSK cells in bone marrow and spleen were significantly increased,meanwhile the proportions of GMP and MEP were increased in bone marrow,and the myeloid/erythroid/megakaryotic cells in spleen were significantly increased.However the proportion of CLP was not significantly changed.1.4 GSDMD KO BMT promoted hematopoietic recovery.The peripheral blood and body weight of GSDMD KO bone-marrow derived recipient mice recovered faster after ionizing radiation.2.Effects of GSDMD on the intestinal ionizing radiation damage2.1 Effects of GSDMD on ionizing radiation damage in animals2.1.1 Intestine act as a target organ for GSDMDGSDMD was highly expressed in liver,spleen,lung and intestine of mice.GSDMD KO has obvious protective effect on intestinal ionizing radiation damage.2.1.2 Survival rate of intestinal ABI improved significantly.GSDMD KO mice did not die within 30 days after ABI,while the mortality rate of wild mice was 50%.GSDMD KO significantly improved the survival rate of ABI.2.1.3 GSDMD KO feces standed in quality and quantity.GSDMD KO mice received ionizing radiation,and fecal quality and quantity did not significantly reduce,no obvious hydration,deformation and other morphological changes in 3 days.2.1.4 GSDMD KO kept the ability of proliferation in intestinal cell.The positive rates of Ki-67 and BrdU of GSDMD KO after ionizing radiation were significantly higher than those of wild-type mice.GSDMD KO inhibited the loss of intestinal cell proliferation ability induced by ionizing radiation.2.2 Effects of GSDMD on ionizing radiation damage in cells2.2.1 Expression of GSDMD increased in intestinal cell lines post radiation.After ionizing radiation,the shear activation of GSDMD in mouse small intestinal epithelial cells MODE-K gradually increased with time,which proved that GSDMD was also involved in the process of ionizing radiation damage in cells.2.2.2 construction of GSDMD KO/OE cell linesThe GSDMD KO cell line was constructed using MODE-K,a mouse small intestinal epithelial cell with high GSDMD expression.The GSDMD OE cell line was constructed using 3T3-L1,a mouse embryonic fibroblast with low GSDMD expression.The GSDMD KO/OE cell line was verified by western blot and qPCR,and the GSDMD KO/OE effect was quite significant.2.1.3 GSDMD KO inhibited cytotoxicity.Ionizing radiation can activate GSDMD to increase LDH and the positive rate of PI,while inhibiting GSDMD can effectively reduce the cell damage caused by radiation.2.1.4 GSDMD KO alleviated cell cycle arrest.GSDMD KO alleviated G2/M phase arrest after ionizing radiation,and GSDMD OE aggravated this phenomenon.3.The preliminary molecular mechanism GSDMD involved in intestinal ionizing radiation damage3.1 Ionizing radiation activated GSDMD and its related pathways.3.1.1 Ionizing radiation activated GSDMD.After ionizing radiation stimulation,GSDMD gradually changed from the extensive distribution of villi to the concentration at the top of villi.Immunofluorescence experiments showed that GSDMD began to activate and concentrate on the cell membrane to form pores after receiving ionizing radiation for 8 h,which also proved that GSDMD participated in the process of ionizing radiation damage in vitro.3.1.2 Ionizing radiation activated GSDMD upstream molecules.Protein levels verified that Caspase-1 and Caspase-11,upstream molecules of GSDMD,were up-regulated after ionizing radiation.Immunohistochemistry of Caspase-11also confirmed the results.3.1.3 Ionizing radiation activated downstream GSDMD molecules.The expression of IL-1?and IL-18 in tissues and cells,GDSMD downstream cytokines,decreased after ionizing radiation,but those in serum and cell supernatant increased,proving that ionizing radiation promoted the release process of IL-1?and IL-18.3.2 Ionizing radiation activated GSDMD through ROS mediation to exert damage effect.3.2.1 GSDMD KO inhibited the decrease of SOD and the increase of MDA after ionizing radiation.After ionizing radiation,SOD in wild-type mice was significantly inhibited,while SOD in GSDMD KO mice was slightly reduced,but there was no significant difference.The MDA level of wild-type mice was significantly increased by about 4 times after irradiation,while that of GSDMD KO mice was not significantly changed.There was a significant difference between wild-type mice and GSDMD KO mice after irradiation.3.2.2 GSDMD KO inhibited the production of ROS and superoxide anions after ionizing radiation.ROS levels were lower in GSDMD KO cells after ionizing radiation than that in control cells,but the opposite effect was observed in GSDMD OE cells.The trend of superoxide anions was consistent with ROS.3.2.3 GSDMD KO inhibited the reduction of mitochondrial membrane potential after ionizing radiation.After irradiation,the mitochondrial membrane potential of GSDMD KO cells decreased less than that of the control group.In contrast,irradiation induced a significant decrease in mitochondrial membrane potential in GSDMD OE cells,while the control cells had a higher mitochondrial membrane potential.3.2.4 Ionizing radiation induced ROS and activated GSDMD.H₂O₂ was used as a ROS positive drug for protein level verification,and the results showed that H₂O₂ stimulation had a similar effect with ionizing radiation stimulation to some extent,and both of them could activate GSDMD related pathways.3.3 Screening of RNA-SEQ differentially expressed genes and validation of Wnt related pathways3.3.1 RNA-SEQ was used to screen differentially expressed genes.RNA-SEQ was used to screen the differentially expressed genes of wild type and GSDMD KO mice after ionizing radiation.A total of 502 differentially expressed genes were obtained,including 209 up-regulated genes and 293 down-regulated genes.GO analysis and KEGG analysis were performed on the differentially expressed genes.3.3.2 Validation of Wnt related pathwaysSome differentially expressed genes were selected for qPCR verification,which was consistent with the RNA-SEQ results.The Wnt related pathways were selected for RNA and protein level verification,proving that GSDMD KO had significant differences in the Wnt pathway,which need to be further studied.ConclusionIn this study,the effects of GSDMD on hematopoiesis and intestinal ionizing radiation damage and the preliminary molecular mechanism were studied.The research conclusions are as follows:1.GSDMD KO can effectively inhibit the hematopoiesis ionizing radiation damage.GSDMD KO can effectively improve the survival rate of irradiated mice,accelerate peripheral blood recovery,and alleviate ionizing radiation damage of bone marrow and spleen.GSDMD KO can increase the number of nucleated cells in hematopoietic organs,increase the number and proportion of LSK,and promote differentiation of myeloid progenitor.The specific mechanism remained to be further studied.2.GSDMD KO can effectively inhibit intestinal ionizing radiation damage.In vivo,GSDMD is highly expressed in the intestinal and can be activated by ionizing radiation.GSDMD KO can effectively alleviate intestinal injury caused by ionizing radiation,improve survival rate of local abdominal irradiation,maintain fecal formation and normal morphology,and inhibit the loss of intestinal proliferation.In vitro,ionizing radiation can induce up-regulation of GSDMD spliceosome.GSDMD KO can slow down LDH generation,reduce the positive rate of PI,and alleviate cell cycle arrest,while GSDMD OE was the opposite.3.Ionizing radiation exerted radiation damage effect on intestinal by activating GSDMD and its related pathways.Ionizing radiation can regulate the activation and distribution of GSDMD,up-regulate the expression of Caspase-1 and Caspase-11 in the upstream molecules of GSDMD,promote the release of IL-1?and IL-18 in the downstream,and exert the effect of ionizing radiation damage.In addition,ionizing radiation can activate GSDMD and its related pathways through the generation of ROS mediated by ionizing radiation.Finally,ionizing radiation may activate GSDMD by regulating Wnt related pathways to exert ionizing radiation damage effect.