| Background:Liver transplantation refers to the only effective therapeutic method of treating end-stage liver disease.Ischemia-reperfusion(I/R)injury is inevitable during liver transplantation;it critically causes graft dysfunction after transplantation.Hepatic I/R injury refers to liver ischemia attributed to liver transplantation,hepatectomy,etc.When liver reperfusion restores blood supply,liver function will not be restored,whereas liver injury will be aggravated,accompanied by cell dysfunction and structural damage.Several studies reported that I/R injury could cause early graft dysfunction after liver transplantation,primary graft nonfunction,ischemic biliary injury,recurrence of liver cancer,as well as evidently longer Intensive Care Unit(ICU)monitoring time after liver transplantation.Moreover,hepatic I/R injury can also induce acute kidney injury and arrhythmia after transplantation.To mitigate liver I/R injury and elevate the success rate of surgery and the survival rate of patients,the existing common methods consist of ischemic pretreatment and drug pretreatment,whereas all these exhibit certain limitations.Accordingly,it is still urgently required to elucidate the development mechanism of liver I/R injury to provide novel targets and ideas for the treatment of liver I/R injury.Global studies have agreed that liver I/R injury is split into two typical stages,namely,ischemia and reperfusion,and it complies with a unique tissue injury mechanism.With the decrease in the availability of ATP at the ischemic phase,ATP-dependent ion channels will begin to fail,cell metabolism will be hindered,anaerobic glycolysis will be activated,and liver cells will die rapidly.Reperfusion injury consists of direct and indirect cytotoxic mechanisms.At this stage,the liver will produce reactive oxygen species(ROS)to stimulate inflammatory response(e.g.,macrophages and neutrophils infiltration)and cytokines production,eventually causing hepatocyte apoptosis and necrosis.As a result excessive inflammation and apoptosis are considered two vital factors affecting liver I/R injury.Thus,how to reduce the inflammatory response and mitigate apoptosis in liver I/R injury is critical to improve the quality of donors and facilitate the prognosis of liver transplantation.TRIM family proteins are of crucial significance in different biological processes(e.g.,cell proliferation and apoptosis,innate immunity and inflammatory response).All TRIM family proteins contain three domains,namely,RING fingers,B-box zinc fingers and coiled-coil domains.TRIM27,as a heterology of TRIM family proteins,was initially reported as a fusion protein of the RET proto-oncogene in 1988.Several studies reported that TRIM27 down-regulated anti-viral and inflammatory responses by inhibiting IKKα/β/s-mediated activation of NF-κB and IFN.Moreover,TRIM27 knockdown induced apoptosis in ovarian cancer cells by up-regulating p-p38.However,whether and how TRIM27 impacts liver I/R injury remains unclear.Accordingly,the role of TRIM27 in liver I/R injury should be studied.It is helpful to clarify the mechanism of liver I/R injury,theoretically underpin the protection of liver I/R injury and provide novel drug targets.Part 1 The correlation of TRIM27 expression in liver I/R injuryObjective:To delve into the expression correlation of TRIM27 in hepatic I/R injury.Methods:1.Detect the correlation of TRIM27 expression in liver I/R injury1.1 Liver tissue samples were collected from patients of liver transplantation before they underwent cold perfusion and before abdominal closure after liver transplantation.The protein and gene expression levels of TRIM27 in liver tissue were detected by Western-blot assay,immunohistochemistry,and RT-PCR.1.2 A mouse liver I/R model was built,and protein and gene expression variations of TRIM27 in liver tissue were detected by Western-blot assay,immunohistochemistry,and RT-PCR.1.3 The model of hepatocyte hypoxia/reoxygenation(H/R)was built,and the expression of TRIM27 was detected by Western-blot assay.Results:In liver transplantation clinical samples,compared with liver tissue samples before patents underwent liver transplantation,the expressions of TRIM27 gene and protein after liver transplantation were considerably down-regulated;the gene and protein expressions of TRIM27 in liver tissue of mice after liver I/R surgery were significantly down-regulated in contrast to those of the sham control group.Besides,the expression of TRIM27 protein in hepatocyte was significantly down-regulated after H/R treatment compared with that of the control group.Conclusion:TRIM27 was significantly down regulated during hepatic I/R injury,suggesting that it may play an important role in hepatic I/R injury.Part 2 The effect of TRIM27 on inflammatory response and apoptosis during liver I/R injuryObjective:To delve into the effect of TRIM27 on inflammatory response and apoptosis during liver I/R injury.Methods:1.Ascertain the effect of TRIM27 on liver injury in mice after I/R surgery1.1 Trim27 knockout(Trim27-KO)mice were constructed,and a liver I/R injury model was built.The knockout efficiency of TRIM27 was ascertained by Western-blot assay;the content of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum of mice were measured with a fully automatic biochemical analyzer;the area of liver necrosis was assessed by hematoxylin-eosin staining(H&E)staining.1.2 Hepatocyte-specific Trim27 transgenic(Trim27-HTG)mice were constructed,and a liver I/R injury model was built.The expression of TRIM27 was detected by Western-blot assay;the levels of ALT and AST in serum of mice were detected with a fully automatic biochemical analyzer;the area of liver tissue necrosis and the degree of necrosis were assessed by H&E staining.2.Detect the regulation of TRIM27 on the inflammation of mouse liver I/R injury2.1 Trim27-KO mice were constructed,and a liver I/R injury model was built.CD11b immunofluorescence was exploited to ascertain the infiltration of inflammatory cells in liver tissue;RT-PCR was performed to detect the expressions of Tnfα,116,Il1b,Ccl2,and Cxcl2 in liver tissue;Western-blot assay was conducted to detect the expression of NF-κB signaling pathway-related molecules.2.2 A TRIM27 knockdown stable transfected cell line was established through lentivirus infection,and a model of hepatocyte H/R was built.RT-PCR and Western-blot assay were performed to detect the knockdown of TRIM27;RT-PCR was conducted to detect the expression of inflammatory factors(e.g.,Tnfα,116,and Il1b).2.3 Trim27-HTG mice were constructed,and a liver I/R injury model was built.CD11b immunofluorescence was performed to test the infiltration of inflammatory cells in liver tissues;RT-PCR was performed to detect the expression of Tnfa,116,Il1b,Ccl2,and Cxcl2 in liver tissue;Western-blot assay was performed to detect the expression of NF-κB signaling pathway-related molecules.2.4 A stable transfected cell line of TRIM27 overexpression was established through lentivirus infection,and a model of hepatocyte H/R was built.The overexpression of TRIM27 was detected by RT-PCR and Western-blot assay;RT-PCR was performed to detect the expression of inflammatory factors(e.g.,Tnfα,Il6,and 111b).3.Detection of TRIM27 on the apoptosis of mouse liver during I/R injury3.1 Trim27-KO mice were established,a liver I/R injury model was built.TUNEL immunofluorescence and c-CASPASE-3 immunohistochemistry were performed to test apoptosis of liver tissue;RT-PCR was performed to detect the expression of apoptosis-related genes(e.g.,Bad,Bax,and Bcl2);Western-blot assay was conducted to detect the expression of apoptosis-related proteins(e.g.,BAX,BAD,BCL2 and c-CASPASE-3).3.2 A TRIM27 knockdown stable transfected cell line was established by lentivirus infection,and a model of hepatocyte H/R was built.RT-PCR was performed to detect the expression of apoptosis-related genes(e.g.,Bax and Bcl2).3.3 Trim27-HTG mice were constructed,and a hepatocyte H/R model was built.TUNEL immunofluorescence and c-CASPASE-3 immunohistochemistry were performed to detect apoptosis of liver tissue;RT-PCR was performed to detect the expression of apoptosis-related genes(e.g.,Bad,Bax,and Bcl2);Western-blot assay was conducted to detect the expression of apoptosis-related proteins(e.g.,BAX,BAD,BCL2 and c-CASPASE-3).4.4 A stable transfected cell line of TRIM27 overexpression was established by lentivirus infection,and a model of hepatocyte H/R was built.RT-PCR was performed to detect the expression of apoptosis-related genes(e.g.,Bax and Bcl2).Results:1.TRIM27 reduced liver injury after liver I/R surgeryCompared with the WT group,the serum aminotransferases(ALT,AST)in mice of Trim27-KO group were significantly up-regulated after liver I/R injury,and the area of liver necrosis was significantly expanded.In contrast to the NTG group,the serum aminotransferases(ALT,AST)in mice of Trim27-HTG group were considerably down-regulated after liver I/R injury,and the area of liver necrosis was significantly narrowed.2.TRIM27 inhibited the inflammatory response during liver I/R injuryCompared with the WT group,the infiltration of CD11b-positive inflammatory cells in liver tissue was evidently facilitated after liver I/R injury in the Trim27-KO group.The expressions of inflammatory factors(e.g.,Tnfa,Il6,Il1b,Ccl2,and Cxcl2)in liver tissue were obviously up-regulated,and the NFκB pathway was activated.Compared with the control group,the expressions of inflammatory factors(e.g.,Tnfα,Il6,Il1b)were considerably up-regulated in hepatocytes of TRIM27 knock-down group after H/R treatment.Compared with the NTG group,the infiltration of CD lib-positive inflammatory cells in liver tissue was obviously hindered after liver I/R injury in the Trim27-HTG group.The expressions of inflammatory factors(e.g.,Tnfα,116,Il1b,Ccl2,and Cxcl2)in liver tissue were remarkably down-regulated,and the NFκB pathway was inhibited.Compared with the control group,the expressions of inflammatory factors(e.g.,Tnfα,116,and Il1b)were evidently down-regulated in TRIM27 over-expressed hepatocytes after H/R treatment.3.TRIM27 suppressed apoptosis during liver ischemia-reperfusion injuryCompared with the WT group,TUNEL and c-CASPASE-3 positive apoptotic cells in liver tissue were considerably enriched after liver I/R injury in Trim27-KO group.The gene and protein expressions of pro-apoptotic factor(e.g.,BAX,BAD and c-CASPASE3)were obviously up-regulated,and gene and protein expressions of anti-apoptotic factor BCL2 were remarkably down-regulated;in comparison with those of the control group,the gene expression of pro-apoptotic factor Bax was evidently up-regulated after H/R in hepatocytes of the TRIM27 knock-down group,and the gene expression of anti-apoptosis factor Bcl2 was considerably down-regulated;in contrast to those of the NTG group,TUNEL and c-CASPASE-3 positive apoptotic cells in liver tissue were obviously reduced after liver I/R injury in the Trim27-HTG group,and the gene and protein expressions of pro-apoptotic factors(e.g.,BAX,BAD,and c-CASPASE-3)were remarkably down-regulatedand the gene and protein expressions of anti-apoptotic factor BCL2 were evidently up-regulated;compared with those of the control group,the gene expression of pro-apoptotic factor Bax was considerably down-regulated,and the gene expression of anti-apoptotic factor Bcl2 was obviously up-regulated in TRIM27 over-expressed hepatocytes after H/R treatment.Conclusion:TRIM27 could inhibit the inflammatory response and apoptosis during hepatic I/R injury,thereby mitigating the liver I/R injuryPart 3 The mechanism of TRIM27 regulating hepatic I/R injury through TAK1-JNK/p38 signal axisObjective:To elucidate the molecular mechanism of TRIM27 regulating hepatic I/R injury via TAK1-JNK/p38 signal axis.Methods:1.Explor the mechanism of TRIM27 regulating liver I/R injury by high-throughput sequencing1.1 Detection of differential gene variations in liver tissue in WT and Trim27-KO mice after liver I/R surgery by high-throughput sequencing;analysis of enriched differential gene-related signal pathway variations after enrichment of Trim27 knockout by Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis;detection of the expression variations of inflammatory genes involved in Gene Set Enrichment Analysis(GSEA)pathway by heat map.1.2 Trim27-KO and WT mice were recruited to build a liver I/R injury model.The variations of total protein and phosphorylated proteins of MAPK pathway related molecules(e.g.,ASK1,TAK1,ERK,JNK,and p38)were ascertained by Western-blot assay;TRIM27 knockdown stable transfected cell lines were constructed by lentivirus infection,and hepatocyte H/R model was built.The variations of total protein and phosphorylated protein of MAPK pathway related molecules(e.g.,TAK1,JNK and p38)were detected by Western-blot assay.1.3 Trim27-HTG and NTG mice were recruited to build a liver I/R injury model.Western-blot assay was performed to detect the variations of total proteins and phosphorylated proteins of related molecules(e.g.,ASK1,TAK1,ERK,JNK,and p38)of MAPK pathways;TRIM27 overexpression stable transfected cell lines were established by lentivirus infection,and hepatocyte H/R model was built.The variations of total protein and phosphorylated protein of MAPK pathway related molecules(e.g.,TAK1,JNK and p38)were detected by Western-blot assay.2.Detect whether the regulation of TRIM27 on hepatic I/R injury depends on TAK12.1 A TRIM27 knockdown stable transfected cell line was established through lentivirus infection,and a model of hepatocyte H/R was built addition of NG25,an inhibitor of TAK1.RT-PCR was conducted to detect the expression of inflammatory factors(e.g.,Tnfα,Il6,and Il1b)and the expression of apoptosis-related genes(e.g.,Bax and Bcl2).2.2 By performing cell immunofluorescence,the colocalization of TRIM27 and TAK1 was identified under a laser confocal microscope;the interaction between TRIM27 and TAK1 was identified by co-immunoprecipitation experiment;the direct interaction between TRIM27 and TAK1 was ascertained by GST-pulldown experiment;the interacting domain of TRIM27 and TAK1 was detected by mapping experiment.3.Detect the effect of TAB2/3 in TRIM27-TAK1 mediated liver I/R injury3.1 Myc-Ub,HA-TAK1 and Flag-TRIM27 plasmids were transfected in HEK293T cells,and the effect of TRIM27 on the ubiquitination level of TAK1 was ascertained.3.2 The interaction between TRIM27 and TAB2/3 was ascertained by co-immunoprecipitation experiments.3.3 Gradient transfection of Myc-TRIM27,and HA-TAB2 or Flag-TAB3 in HEK293T cells to ascertain the effect of TRIM27 on the stability of TAB2/3 protein.3.4 Gradiently transfection of Myc-TRIM27,and HA-TAB2 or Flag-TAB3 in HEK293T cells,and addition of DMSO,MG132,and CQ,respectively,to ascertain the approach of TRIM27 affecting the stability of TAB2/3 protein.3.5 The effects of TRIM27 on TAB2/3-mediated TAK1 ubiquitination activation in HEK293T cells were ascertained,as well as the effect of TRIM27 in TAB2/3-mediated TAK1 phosphorylation activation in hepatocytes after H/R treatment.Results:1.TRIM27 suppressed TAK1-JNK/p38 signaling pathway activation during liver I/R injuryAfter liver I/R injury in Trim27-KO mice,the MAPK signal pathway was remarkably enriched,and ASK1 and TAK1 expressions were evidently up-regulated.Compared with the WT group,the expression of p-TAK1,p-JNK,and p-p38 protein in liver tissues were considerably up-regulated after I/R injury in mice of the Trim27-KO group;After H/R treatment was performed,p-TAK1,p-JNK,and p-p38 protein expressions were evidently up-regulated in contrast to the control group.Compared with the NTG group,the protein expressions of p-TAK1,p-JNK,and p-p38 in liver tissue of the Trim27-HTG group exhibited considerable down-regulation after liver I/R injury;After H/R treatment,the protein expressions of p-TAK1,p-JNK,and p-p38 were evidently down-regulated.2.The regulatory effect of TRIM27 on hepatic I/R injury depended on TAK1Knockdown of TRIM27 significantly promoted the inflammatory response and apoptosis in hepatocytes after H/R.The addition of TAK1 inhibitor counteracted the effects of TRIM27 knockdown on inflammation and apoptosis.TRIM27 and TAK1 were co-localized in the hepatocyte cytoplasm;as demonstrated from the results of Co-IP experiments,TRIM27 and TAK1 banded together;GST-pulldown experiments confirmed that TRIM27 and TAK1 could directly interact with each other.Mapping experiments demonstrated that the interaction between 133-513 amino acid sequence of TRIM27 and 1-480 amino acid sequence of TAK1.3.TRIM27 inhibited TAK1 activation by degrading TAB2/3TRIM27 inhibited the K630 ubiquitination activation of TAK1;TRIM27 respectively interacted with TAB2 and TAB3;TRIM27 degraded TAB2/3 via the lysosomal pathway;TRIM27 inhibited TAB2/3-mediated ubiquitination and phosphorylation activation of TAK1.Conclusion:TRIM27 inhibits TAK1-JNK/p38 signal pathway by down-regulating TAB2/3 to mitigate hepatic I/R injury. |
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