The Role And Molecular Mechanism Of E3 Ubiquitin Ligase TRIM65 In Intestinal Ischemia And Reperfusion Injury

Objective Intestinal ischemia-reperfusion injury(II/R)is the injury of intestinal tissue and a series of distant organs caused by the interruption of blood oxygen supply and subsequent tissue reperfusion.In severe cases,it can develop into MODS,in which apoptosis is an important part of the injury process.TRIM65 can regulate apoptosis in some diseases through E3 ubiquitin ligase activity,and TOX4 has been screened as one of the substrate proteins of TRIM65.Therefore,this study will focus on the following four aspects: Firstly,we will reveal the role of TRIM65 in II/R;Then,clarify the interaction between TRIM65 and TOX4 and the molecular mechanism of ubiquitination modification of TOX4 by TRIM65;Finally,verify that TRIM65 targets TOX4 to regulate intestinal epithelial cells apoptosis in intestinal ischemia-reperfusion injury through its E3 ubiquitin ligase activity.This study aims to reveal the mechanism of E3 ubiquitin ligase TRIM65 in intestinal ischemia-reperfusion injury from the molecular,cellular,and animal levels,which will provide a new target for the treatment of II/R injury.Method 1.C57BL/6J wild-type mice were selected to construct an intestinal ischemiareperfusion model,and the extent of intestinal injury was assessed by HE staining,and the expression of TRIM65 in mouse intestinal tissues was detected by Western blot and IHC staining;CACO-2 cells and IEC-6 cells were selected to construct a cellular hypoxia-reoxygenation model,and the expression of TRIM65 in CACO-2 cells and IEC-6 cells was detected by Western blot.2.TRIM65 knockout mice were obtained by gene knockout technique to establish intestinal ischemia-reperfusion model.Intestine was collected for HE staining to observe the degree of intestinal injury.Western blot and IHC staining were used to detect the expression of TRIM65,BAX and BCL-2.TUNEL fluorescence staining was used to analyze the changes of tissue apoptosis.Serum was collected to detect the changes of I-FABP,DAO,TNF-α,IL-6 and IL-1β by ELISA.IEC-6 cells with high and low TRIM65 expression were obtained by lentiviral infection technique to establish a cellular hypoxia-reoxygenation model,the cell samples were collected to detect the expression of TRIM65,BAX and BCL-2 by western blot,and the apoptosis level was detected by Annexin V-Alexa Fluor647/PI apoptosis kit.3.The presence of interaction between TRIM65 and TOX4 was detected by immunoprecipitation and GST Pull-down assay,the location of the TRIM65-TOX4 interaction fragment was detected by immunoprecipitation,and the colocalization of TRIM65 and TOX4 in cells was observed by cellular immunofluorescence.4.Using CHX and MG132 to treat IEC-6 cells and 293 T cells,Western blot was performed to detect TOX4 protein degradation in wild-type cells and TRIM65 high expressing cells;ubiquitination assay was performed to determine the ubiquitination effect of TRIM65 on TOX4 and reveal the type of ubiquitination modification;Western blot was performed to detect the protein expression of BAX and BCL-2 in the cells when TOX4 reverted to high expression;the expression of TOX4 in the intestinal tissues of four groups of mice,WT + I/R,WT + Sham,KO + I/R and KO + Sham,was detected by IHC staining.Result 1.The results of HE staining showed that the degree of intestinal injury in the group of intestinal ischemia-reperfusion for 6 h was significantly aggravated.The results of IHC staining and western blot showed that the protein expression of TRIM65 was decreased in intestinal tissues after 6 hours of intestinal ischemia-reperfusion;in CACO-2 cells after 1 hour of cellular hypoxia-reperfusion;and in IEC-6 cells after 4 hours of cellular hypoxiareperfusion.2.Compared with wild type mice,HE staining in TRIM65 knockout mice showed that intestinal injury was aggravated,IHC staining and western blot results showed an increase in the ratio of BAX to BCL-2 protein expression,and TUNEL staining showed increased apoptosis.ELISA results showed elevated serum levels of I-FABP,DAO,and inflammatory factors.Western blot results showed that compared with the control IEC-6 cells,the ratio of BAX to BCL-2 protein expression decreased in TRIM65 high expressing IEC-6 cells after hypoxia reoxygenation,and the ratio of BAX to BCL-2 protein expression increased in TRIM65 low expressing IEC-6 cells;the results of apoptosis kit showed that the degree of apoptosis decreased in TRIM65 high expressing cells.3.GST Pull-down and immunoprecipitation results showed a direct interaction between TRIM65 and TOX4,;amino acid sequences 1-300 of TOX4 were bound to the CC and SPRY regions of TRIM65,and cellular immunofluorescence results showed co-localization of TRIM65 and TOX4 in cells,and enhanced intensity of TOX4 and TRIM65 co-localization in IEC-6 cells of MG132-treated group compared with the control group.4.The results of protein degradation half-life assay showed that high expression of TRIM65 accelerated the degradation of TOX4 protein in IEC-6 cells,while the protein degradation of TOX4 was inhibited after treatment with MG132;the results of immunoprecipitation showed that TRIM65 ubiquitinated TOX4,and the type of ubiquitination was the ubiquitination of K48 junction site;the results of Western blot assay of cellular reversion to high TOX4 expression and IHC staining of intestinal tissues showed that TRIM65-TOX4 was involved in the apoptotic process of intestinal ischemia-reperfusion cells.Conclusion Our results suggest that TRIM65 knockdown promotes apoptosis and aggravates intestinal tissue injury and inflammation during II/R,and TRIM65 promotes ubiquitinated degradation of TOX4 via E3 ubiquitin ligase activity as one of its possible molecular mechanisms of action.