| PART ?:CHANGES OF OTUD4 EXPRESSION LEVEL IN VIVO AND IN VITRO MODELS OF LIVER ISCHEMIA-REPERFUSIONObjective:Explore the temporal pattern of OTUD4 expression in vivo and in vitro models of liver ischemia-reperfusion,and measure the K63 ubiquitination level of TRAF6 during NF-?B activation.Detection of K63 ubiquitination of TRAF6 during NF-?B activation.Methods:1.Construction of mouse liver ischemia and reperfusion(IR)model.1)Mice were randomly divided into four groups: sham,I1R3,I1R6,I1R9("I" stands for ischemic duration and "R" stands for reperfusion duration).2.ELISA to detect the expression of IL-1? in serum of mice at different reperfusion times.3.Construction of cell hypoxia reoxygenation(HR)model.Randomly divided into six groups: control group(Control),H9R1,H9R2,H9R3,H9R6,H9R9("H" represents the duration of hypoxia and "R" represents the duration of reoxygenation).4.Western-blot to detect the expression level of OTUD4 and related proteins in IR model and HR model at each time point.5.Detection of TRAF6 ubiquitination by immunoprecipitation and western blot.Results:1.The mouse IR model was successfully established,the liver was pink before ischemia,and the liver was grey-white after non-invasive vascular clamp blocked 70% of blood supply.2.Western-blot results showed that compared with the Sham group,the level of OTUD4 expression in the IR model was down-regulated,and gradually decreased with the reperfusion time,the difference was statistically significant(p <0.05).3.ELISA results showed that compared with the Sham group,the level of IL-1? expression in the IR model was up-regulated and gradually increased with the reperfusion time,the difference was statistically significant(p <0.05).4.Western-blot results showed that: compared with the control group,the expression level of p-NF-?B p65 protein in the cellular HR model was up-regulated and gradually increased with prolonged reoxygenation time;and the expression levels of OTUD4 and I?B-? protein It wasdown-regulated and gradually decreased with prolonged reoxygenation time.The difference was statistically significant(p <0.05).5.The results of immunoprecipitation and western blot showed that compared with the control group,the TRAF6 protein and its K63 ubiquitination level in the cellular HR model were up-regulated and gradually increased with the reoxygenation time,the difference was statistically significant(p < 0.05).Conclusion:The expression levels of OTUD4 in mouse IR model and cell HR model decreased with increasing reperfusion / reoxygenation time;while p-NF-?B p65,TRAF6 protein and its K63 ubiquitination level increased with reperfusion / reoxygenation time and up-regulation.PART ?:THE ROLE AND MECHANISM OF OTUD4 IN AN IN VITRO MODEL OF LIVER ISCHEMIA-REPERFUSIONObjective:To explore the relationship between OTUD4 and K63 ubiquitination of TRAF6 and activation of NF-?B,and to preliminary explore the role and mechanism of OTUD4 in an in vitro model of liver ischemia-reperfusion.Methods:1.Co-immunoprecipitation and western blot detection of the interaction of TRAF6 and OTUD4.2.Construction of OTUD4 over-expressing lentivirus,and selection of stable strains after infection of cells.3.After infected cells with Lentivirus transfect-OTUD4overexpression(LV-OTUD4)and for 72 hours,the cells were divided into three groups: non-transfected cells were used as the control group,negative control group(NC),LV-OTUD4 group.Western-blot was used to detect the expression levels of OTUD4 and TRAF6 to verify the effect of OTUD4 overexpression.4.After infected cells with Lentivirus transfect-OTUD4overexpression(LV-OTUD4)for 72 hours,the cells were divided into four groups: cells without hypoxia and reoxygenation were used as the controlgroup(Control),and non-transfected cells treated with hypoxia and reoxygenation were the HR group,hypoxia and reoxygenation treated negative control cell group(HR + NC),hypoxia and reoxygenation treated OTUD4 overexpressing cell group(HR + LV-OTUD4).The effect of OTUD4 overexpression on TRAF6 ubiquitination was verified by immunoprecipitation and western blot.5.After infected cells with Lentivirus transfect-OTUD4overexpression(LV-OTUD4)for 72 hours,the cells were divided into four groups: Control group,HR group,HR + NC group,and HR + LV-OTUD4 group.Western-blot was used to detect the effect of overexpression of OTUD4 on NF-?B activation.6.After infected cells with Lentivirus transfect-OTUD4overexpression(LV-OTUD4)for 72 hours,the cells were divided into four groups: Control group,HR group,HR + NC group,and HR + LV-OTUD4 group.Cellular immunofluorescence was used to detect the effect of OTUD4 overexpression on the transport of NF-?B p65 into the nucleus.7.After infected cells with Lentivirus transfect-OTUD4overexpression(LV-OTUD4)for 72 hours,the cells were divided into four groups: Control group,HR group,HR + NC group,and HR + LV-OTUD4 group.The effect of inflammatory factor IL-1? expression in the cell supernatant was detected by ELISA.Results:1.The results of co-immunoprecipitation and western blot showed that OTUD4 protein and TRAF6 protein were directly bound in RAW264.7cells.2.Successfully infected cells with OTUD4 overexpression lentivirus and obtained stable strains.3.Western-blot results showed that there was no statistical difference in the expression of OTUD4 and TRAF6 protein between the control group and the NC group;there was no statistical difference in the expression of TRAF6 protein between the LV-OTUD4 group and the control group,but the expression of OTUD4 protein was significantly increased(P <0.05).4.The results of immunoprecipitation and western blot showed that compared with the control group,the TRAF6 protein and its K63 ubiquitination level were increased in the HR group(p <0.05).Compared with the HR group,the TRF6 protein and its K63 ubiquitination level were not statistically different in the HR + NC group;compared with the HR group,the TRF6 protein expression level in the HR + LV-OTUD4 group was not statistically different from the HR group,but TRAF6 Expression of K63 ubiquitination was down-regulated(p <0.05).5.Western-blot results showed that compared with the control group,the expression levels of I?B-? and OTUD4 protein were down-regulated,while the expression levels of p-NF-?B p65 and TNF-? protein wereup-regulated(p <0.05).Compared with the HR group,the TNF-?,p-NF-?B p65,I?B-?,and OTUD4 protein expression levels were not statistically different in the HR + NC group;compared with the HR group,the OTUD4 and OTUD4 in the HR + LV-OTUD4 group were not significantly different.I?B? protein expression levels were up-regulated,while TNF-? and p-NF-?B p65 protein expression levels were down-regulated(p <0.05).6.Cell immunofluorescence results showed that NF-?B p65 in the control group was mainly distributed outside the nucleus.Compared with the control group,the NF-?B p65 nucleation rate increased in the HR group,and a large amount of red fluorescence was observed in the nucleus.Compared with the HR group,the nuclear entry of NF-?B p65 in the HR +NC group was not significantly different,while the nuclear entry rate of the HR + LV-OTUD4 group was reduced,and only a small amount of red fluorescence was observed in the nucleus.7.ELISA results showed that compared with the control group,the expression level of IL-1? in the HR group was up-regulated(p <0.05).Compared with the HR group,the expression of IL-1? in the HR + NC group was not statistically different;compared with the HR group,the expression of IL-1? in the HR + LV-OTUD4 group was down-regulated(p<0.05).Conclusion:Cell experiments have confirmed that over-expression of OTUD4 hasno effect on the expression level of TRAF6 protein,but it can negatively regulate the activation of NF-?B and inhibit the release of inflammatory factors by removing the K63 polyubiquitin chain of TRAF6.PART ?:THE ROLE OF OTUD4 IN MICE LIVER ISCHEMIA-REPERFUSION MODELObjective:To explore the role of OTUD4 in liver ischemia-reperfusion injury in an in vivo model.Methods:1.Construction of mice IR model.The mice were randomly divided into four groups: sham,IR,IR + NC,and IR + LV-OTUD4.Two weeks before the IR model was established in mice,no-load lentivirus and OTUD4 over-expressing lentivirus were injected into the corresponding groups through the tail vein of C57 BL / 6J mice,respectively.2.Western-blot was used to detect the effect of OTUD4 overexpression on NF-?B activation.3.ELISA was used to detect the effect of IL-1? expression in the serum of mice.4.Detection of AST and ALT levels in mouse serum by microplate method.5.Liver tissue HE staining to detect liver tissue damage.Results:1.Western-blot results showed that compared with the sham group,the expression levels of I?B-? and OTUD4 protein were down-regulated inIR group,while the expression levels of p-NF-?B p65 and TNF-? protein were up-regulated(p <0.05).Compared with IR group,the expression levels of TNF-?,p-NF-?B p65,I?B-? and OTUD4 protein in IR + NC group were not statistically different;compared with IR group,OTUD4 and I?B? protein in IR + LV-OTUD4 group The expression levels were up-regulated,while the expression levels of TNF-? and p-NF-?B p65 proteins were down-regulated(p <0.05).2.ELISA results showed that compared with the sham group,the IL-1? expression level in the IR group was up-regulated(p <0.05).Compared with the IR group,the IL-1? expression level in the IR + NC group was not statistically different;compared with the IR group,the IL-1?expression level in the IR + LV-OTUD4 group was down-regulated(p<0.05).3.The results of microplate detection of mouse serum showed that compared with the sham group,the expression levels of AST and ALT in the IR group were up-regulated(p <0.05).Compared with IR group,AST and ALT expression levels were not statistically different in IR + NC group;compared with IR group,AST and ALT expression levels were down-regulated in HR + LV-OTUD4 group(p <0.05).4.The results of liver HE staining showed that the liver structure of the mice in the sham group was normal,the morphology and size of liver cells were normal,and there were no pathological features such asinflammatory cell infiltration or ballooning.Compared with the sham group,the IR group had structural disorder,cell necrosis,and inflammatory infiltration,and the SUZUKI score increased(p <0.05).Compared with the IR group,there was no significant difference in the degree of cell damage and inflammatory infiltration in the IR + NC group,and there was no statistical difference in SUZUKI score.Compared with the IR group,the range of cell necrosis,inflammatory infiltration and the vacuole formation was reduced and the SUZUKI score was reduced,the difference was statistically significant(p <0.05).Conclusion:In vivo experiments in mice have confirmed that over-expression of OTUD4 can negatively regulate the activation of NF-?B,inhibit the release of inflammatory factors,and reduce liver tissue damage caused by ischemia-reperfusion. |
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