The Effect And Mechanism Of LncRNA NBR2 Regulating Endothelial Pyroptosis By Targeting GSDMD In Sepsis

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The effect and mechanism of GSDMD-mediated endothelial pyroptosis on vascular endothelial injury in sepsisOBJECTIVE: To investigate the effect and mechanism of GSDMD-mediated endothelial pyroptosis on vascular endothelial injury in sepsis.METHODS: Endothelial cells EA.hy926 were transfected with LPS(tLPS)to induce endothelial pyroptosis.Endothelial cells were transfected with empty or GSDMD shRNA,and then the morphological changes of pyroptic cells were measured by light microscopy,LDH release was tested by lactate dehydrogenase(LDH)cytotoxicity assay kit,IL-1? was tested by ELISA,and pyroptosis was tested by flow cytometry.RT-qPCR was used to detecte the expression of inflammatory Caspase and GSDMD mRNA,Western Blot or immunofluorescence was used to detecte the expression of inflammatory Caspase and GSDMD protein.Adeno-associated virus containing sh Gsdmd vector was injected via tail vein to interfere with Gsdmd expression in mice,and a mouse model of sepsis was constructed by cecum ligation and puncture(CLP).Pulmonary vascular endothelium alterations were detected by transmission electron microscopy,lung wet weight to body weight ratio and Evans blue exudation was measured,protein content in bronchial alveolar lavage fluid(BALF)was detected by BCA assay,plasma level of IL-1?,IL-18 and TNF-? secretion was detected by ELISA,and organ injury was detected by HE staining.RESULTS: tLPS stimulation resulted umbilical vein endothelial cells pyroptosis,significantly enhanced LDH release,dramatically boosted IL-1? secretion,markedly increased expression of inflammatory Caspase and GSDMD mRNA and protein,and significantly increased inflammatory Caspase and GSDMD cleavage.With the GSDMD shRNA treatment,the expression of GSDMD mRNA and protein were significantly reduced in endothelial cell.tLPS-induced endothelial pyroptosis was conspicuous alleviated,LDH release was significantly reduced,and IL-1? secretion was markedly decreased.The conspicuous pyroptosis was observed in pulmonary vascular endothelial cell in septic mice.The pulmonary tissue edema,protein content in BALF and Evans blue exudation was significantly increased in septic mice.The plasma level of inflammatory factors was increased,and the severe injury in various organs was detected in septic mice.While injection of sh Gsdmd AAV significantly alleviated pulmonary vascular endothelial pyroptosis,improved pulmonary tissue edema,reduced protein exudation,improved pulmonary vascular permeability,and decreased plasma IL-1? and IL-18 levels,and alleviated multiple organ damage in septic mice.CONCLUSION: GSDMD-mediated endothelial pyroptosis leads to vascular endothelial injury and multiple organ dysfunction in sepsis.Chapter ? The effect and mechanism of lncRNA NBR2 targeting GSDMD in regulating septic vascular endothelial pyroptosisOBJECTIVE: To investigate the effect and mechanism of lncRNA in sepsis vascular endothelial pyroptosis.METHODS: Gene microarray was used to detect lncRNA and mRNA expression profiles in the blood of sepsis patients,bioinformatics analysis was performed to screen lncRNAs associated with pyroptosis.Umbilical vein endothelial cells EA.hy926 were transfected with LPS(tLPS)to induce endothelial pyroptosis,and RT-qPCR was performed to detect the expression of candidate lncRNAs.The si RNA of candidate lncRNAs were transfected into endothelial cells to interference with candidate lncRNAs expression,NBR2 overexpression and GSDMD interference plasmid were transfected into endothelial cells to modulate the expression of NBR2 and GSDMD,then these endothelial cells were treated with tLPS.The candidate lncRNAs,NBR2 and GSDMD expression were detected by RT-qPCR,and inflammatory Caspase and GSDMD protein expression was tested by Western Blot or immunofluorescence,the cell morphology was measured by light microscope,LDH release was tested by LDH cytotoxicity assay kit,and IL-1? was tested by ELISA.Nuclear and cytoplasmic extraction reagents was used to separate cytoplasm and nucleus,and NBR2 expression was detected by RT-qPCR respectively.The fluorescence in situ hybridization(FISH)was applied to test the NBR2 sub-cellular localization in endothelial cell.RESULTS: Gene co-expression analysis suggested that 12 lncRNAs were associated with pyroptosis,and 4 of those lncRNAs significantly elevated in pyroptic endothelial cells.With the si RNA of these 4 lncRNAs treatment,only in the si NBR2 groups,tLPS-induced endothelial pyroptosis was conspicuous alleviated,LDH release was significantly reduced,and IL-1? secretion was markedly decreased.And the expression of GSDMD mRNA and protein were significantly reduced in endothelial cell with si NBR2 treatment.The NBR2 gene is located on chromosome 17 q21.31,contains 1371 nucleotides,contains 3 splice isoforms,5 exons,an open reading frame,and without protein-coding ability.The NBR2 expression was markedly enhanced by tLPS stimulation,with a significant increased expression in nuclear.Overexpression of NBR2 promoted GSDMD mRNA and protein expression,and promoted endothelial pyroptosis,LDH release,and IL-1? secretion,while sh GSDMD blocked the effect of NBR2 on promoting endothelial pyroptosis.CONCLUSIONS: lncRNA NBR2 targets and regulates GSDMD expression to mediates septic vascular endothelial pyroptosis.Chapter ? Mechanism of lncRNA NBR2-targeted regulation of GSDMD expressionOBJECTIVE: To investigate the molecular mechanism of NBR2-targeted regulation of GSDMD expression.METHODS: Firstly,the NBR2-bound proteins were captured by Chromatin Isolation by RNA Purification(ChIRP),and the key NBR2-bound protein was detected by liquid-mass tandem mass spectrometry(LC-MS/MS)and HNRNPK was verified by Western Blot.The interaction between NBR2 and HNRNPK was verified by RNA binding protein immunoprecipitation assay(RIP)combined with RT-qPCR.Then,endothelial cells were infected with lentivirus containing sh HNRNPK vector,and stimulated with tLPS.The expression of NBR2,HNRNPK and GSDMD mRNA was detected by RT-qPCR,and the expression of HNRNPK and GSDMD protein was detected by Western Blot or immunofluorescence.The cell morphology was measured by light microscope,LDH release was tested by LDH cytotoxicity assay kit,and IL-1?was tested by ELISA.Then,chromatin immunoprecipitation(Ch IP)combined with qPCR was used to detect the interaction between HNRNPK protein and GSDMD gene,and Dual-luciferase reporter gene assay was used to test the regulatory effect of HNRNPK on GSDMD gene transcription.Finally,endothelial cells infected with sh HNRNPK lentivirus were stimulated with tLPS,and the subcellular distribution and co-localization of NBR2 and HNRNPK protein were detected by FISH and immunofluorescence.Nuclear and cytoplasmic extraction reagents was used to separate cytoplasm and nucleus,and NBR2 expression was measured by RT-qPCR,HNRNPK protein expression detected by Western Blot respectively.RESULTS: ChIRP-MS detected 42 proteins that may bind to NBR2.Considering the ability binding GSDMD gene of candidate proteins,we found that HNRNPK protein may bind to both NBR2 and GSDMD gene.ChIRP-Western Blot combined with RIPRT-qPCR results bi-directionally confirmed the HNRNPK and NBR2 interaction.TLPS stimulation led to endothelial pyroptosis,increased LDH release,increased IL-1?secretion and increased GSDMD expression,whereas sh HNRNPK impaired endothelial pyroptosis,decreased LDH release,abrogated IL-1? secretion and alleviated GSDMD expression,abrogated the effect of NBR2 on promoting GSDMD expression and endothelial pyroptosis.Ch IP-qPCR results showed that HNRNPK interacted with GSDMD gene.Dual-luciferase reporter gene indicated that HNRNPK bound to GSDMD gene to promote its' transcription and enhance expression.Co-localization experiments showed that tLPS stimulated the intranuclear translocation of NBR2 and HNRNPK complex.The sh HNRNPK reduced intranuclear translocation of NBR2.Nucleoplasmic separation assay results also confirmed that tLPS stimulated the intranuclear translocation of NBR2 and HNRNPK,sh HNRNPK interfered with HNRNPK expression,and reduced intranuclear translocation of NBR2.CONCLUSION: lncRNA NBR2 via recruiting and binding HNRNPK protein,translocating into the nucleus to target and regulate GSDMD expression,eventually mediate tLPS-induced endothelial pyroptosis.