Effect Of Trim58 Gene Knockout On Radiation-induced Intestinal Injury In Mice

The intestine is one of the most vulnerable organs of the human body to radiation damage.Intestinal inflammation after high doses of systemic radiation is a main cause for the death of radiation therapy.Tripartite motif containing 58（TRIM 58）plays a role in the early stages of drug-induced enteritis in mice by reducing intestinal mucosal damage during acute stress,but whether TRIM58 plays a role in radiation-induced intestinal inflammation is unclear.In this study,Trim58 knockout mice were established.Trim58 wild-type(Trim58^+/+)mice and Trim58 knockout(Trim58^-/-)mice were subjected to total body irradiation（TBI）to observe the survival rate and weight changes of mice.The weight of thymus and spleen were recorded.The length of small intestinal villi and crypts were calculated after HE staining.Apoptosis of small intestinal epithelial cells were detected by TUNEL.The proliferation capacity of small intestinal cells was observed by immunohistochemistry.Peripheral blood T cells and B cells were analyzed by flow cytometry.NF-κB p65 expression levels were detected by Western Blot.Relevant signaling pathways were analyzed by transcriptome sequencing and verified by q PCR.The main results are as follows:1.Establishment of Trim58 knockout miceExon 2～5 of Trim58 were knocked out by CRISPR/Cas9 in C57BL/6 mice.Compared with wild-type mice,there was no obvious difference in the reproductive ability and testicular tissue morphology of Trim58^-/-mice.2.Effect of Trim58 knockout on radiation-induced intestinal injury in miceAfter TBI,the survival rate,spleen and thymus weight of Trim58^-/-mice were significantly higher than those of Trim58^+/+mice.The villi in small intestinal of Trim58^+/+mice became shorter and their recovery were slower.The intestinal damage of Trim58^-/-mice was lighter than that of Trim58^+/+mice.A large number of apoptotic cells appeared in the intestinal villi of Trim58^+/+mice.After 3 hours of TBI,the apoptosis rate of small intestinal cells in Trim58^-/-mice was significantly less than that of Trim58^+/+mice.At the same time,the number of Ki67⁺cells,Lysozyme⁺cells and Villin⁺cells in Trim58^-/-were significantly higher than that of Trim58^+/+mice.Trim58^-/-mice were found to have a significantly higher proportion of T lymphocytes in the peripheral blood after TBI than Trim58^+/+mice.Compared with Trim58^-/-mice,the NF-κB p65 protein in the cytoplasm of Trim58^+/+mice decreased after TBI,while increased significantly in the nucleus.The m RNA expression levels of the downstream genes IL-6 and TNF-αof the NF-κB pathway were increased.3.Transcriptome sequencing analysisTo study the regulatory network of Trim58 in radiation-induced intestinal injury,transcriptome sequencing was performed on small intestinal tissues of Trim58^+/+and Trim58^-/-mice at day 0 and day3 of TBI.Compared with Trim58^+/+mice,1369 differentially expressed genes were identified in the small intestine of Trim58^-/-mice,of which 591 were downregulated and 778 were upregulated.The upregulated genes were enriched in the oxidative phosphorylation pathway.However,after 3 days of radiation exposure,the genes related to the oxidative phosphorylation signaling pathway in the small intestine of Trim58^-/-mice were significantly downregulated compared with Trim58^+/+mice.This study revealed that absence of Trim58 had no effect on fertility of mice.However,compared with Trim58^+/+mice,Trim58 knockout reduced the radiation-induced intestinal injury and immune organ damage caused by TBI,slowed down the death rate of mice.Trim58knockout protected the proliferation ability of mouse intestinal stem cells,maintained the number of peripheral blood T lymphocytes.inhibited the activation of NF-κB p65 signaling pathway.Transcriptome sequencing analysis revealed that absence of Trim58 played a role in protecting intestinal cells by inhibiting excessive activation of oxidative phosphorylation signaling pathway.