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The Role Of Gasdermin D Inhibitor Necrosulfonamide In Acute Liver Failure

Posted on:2021-09-27Degree:MasterType:Thesis
Country:ChinaCandidate:Y L WuFull Text:PDF
GTID:2494306128469894Subject:Internal medicine (digestive)
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Objective: Acute liver failure(ALF)is a critical disease characterized by the loss of liver function and high mortality.There is no specific cure for ALF.Pyroptosis,with Gasdermin D(GSDMD)being the executioner,is involved in the pathophysiological process of immune dysregulation in ALF.The role of GSDMD inhibitor in ALF has not been reported.This study aimed to explore possible pathways involved in the pathogenesis of ALF by performing bioinformatics analysis on Gene Expression Omnibus database(GEO)related ALF datasets.Then,this study predicted the structure and modification of GSDMD protein by bioinformatics analysis.Finally,this study investigated activation of canonical and non-canonical pyroptosis pathways and the role of GSDMD inhibitor necrosulfonamide(NSA)in ALF mouse model.Methods: 1.We used limma package to screen differentially expressed genes(DEGs)between ALF and normal groups in GEO;The Database for Annotation,Visualization and Integrated Discovery(DAVID)was used to perform Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.2.Homo sapiens GSDMD gene constructed vector was established.Then,this study performed homology analysis and post-translational modification prediction for this protein.3.ALF model was established by lipopolysaccharide/D-galactosamine(LPS/D-Gal N)challenged in C57BL/6J mice.Mice were randomized to divided into 4 groups according to the random number table method: normal group(Control group),acute liver failure group(ALF group),dimethyl sulfoxide group(DMSO group)and NSA intervention group(NSA group).The survival rate was monitored.Liver damage were determined by hematoxylin and eosin(HE)and serum alanine aminotransferase(ALT).Possible mechanisms involved were explored by quantitative real-time PCR,western blot and enzyme-linked immunosorbent assay(ELISA).Results: 1.GEO database related datasets suggested that CASP1(P<0.001),CASP5(P<0.005)and IL-18(P<0.001)up-regulated in ALF.BP analysis of DEGs indicated DEGs mainly enriched in immune response,inflammatory response,chemotaxis,extracellular matrix organization and cell adhesion.KEGG pathways were mainly enriched in phagosome,staphylococcus aureus infection and cell adhesion molecules(CAMs)pathway.2.Homologous analysis suggested high homology between human GSDMD and mouse Gsdmd(70.04%).There were 83 phosphorylation sites of human recombinant GSDMD,and there were 23 possible antigenic peptides.3.In vivo,ALF group all died within 10h;24h survival rate of mice treated with NSA was 60%,which significantly improved(P=0.0001).ALT level was significant decreased in NSA treatment group compared with ALF group(232.0 U/L vs.468.3 U/L;P<0.005).Serum IL-1β level examined by ELISA was significant decreased in NSA treatment group compared with ALF group(55.63 pg/m L vs.110.9 pg/m L;P<0.01).The liver injury features in histology were attenuated in NSA treatment group(P<0.005).The m RNA levels of the upstream molecules of Gsdmd,Casp1 and Casp11,increased in ALF(Casp1,P<0.05;Casp11,P<0.01)while significantly decreased after NSA pretreatment(Casp1,P<0.05;Casp11,P<0.05).Il-1β gene expression decreased after NSA treatment compared with ALF group(P<0.05).The protein level of GSDMD,as well as NACHT LRR and PYD domains-containing protein 3(NLRP3),cleaved caspase-1,cleaved caspase-11,and IL-1β was higher in ALF than control(GSDMD,P<0.0001;NLRP3,P<0.005;cleaved caspase-1,P<0.0001;cleaved caspase-11,P<0.001;IL-1β,P<0.005),and significantly decreased after NSA pretreatment(GSDMD,P<0.01;NLRP3,P<0.05;cleaved caspase-1,P<0.05;cleaved caspase-11,P<0.005;IL-1β,P<0.01).Conclusion: Pyroptosis is activated in ALF.Homologous analysis suggested high homology between human GSDMD and mouse Gsdmd.NSA might alleviate ALF via pyroptosis pathway.
Keywords/Search Tags:Acute liver failure, Pyroptosis, GSDMD, Necrosulfonamide, Bioinformatics
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