Glycyrrhizin Inhibit Lps Induced Macrophage Expression And Release Of Hmgb1 Experimental Research

Objective：HMGB1is a advanced stage inflammatory cytokine that leads theinflamation cascade amplification in sepsis.It palys a key role in the onsetand development of sepsis.Targeting it will gain enough time and benefits inthe treatment of sepsis.Glycyrrhizin,which is extracted from licoriceroots,has been shown to express anti-inflammatory,anti-viral and immuneregulatory activies,whereas the potential effect in the treatment of sepsis israrely reported.We use1ipopolysaccharide to stimulate RAW264.7cells,andthen survey the inhibition effect of glycyrrhizin towards the release andexpression of HMGB1which induced by LPS from RAW264.7cells,toexplore the treatment effect of glycyrrhizin in the sepsis.Methods：The RAW264.7cells was stimulated with100μg/L LPS and100μg/LLPS+1mmol/L GL respectively,culture cells for0,4,8,12,16,20,24and48h.The cell culture supernatant was collected,in which HMGB1proteinwas determined by ELISA.After the total RNA of the cultured cells being extracted,the HMGB1mRNA was determined by Real-time PCR.After thecytoplasm and nucleus protein being extracted,the contents of HMGB1protein was detected by western blot.The localization of HMGB1and it’scontent in cells were observed by Immunofluorescence/Laser confocalmicroscopy.Results：The expression of HMGB1mRNA increased significantly from8to48h after LPS stimulation,the expression of HMGB1mRNA increasedsignificantly from8to16h after LPS+GL stimulation(P<0.01).Theexpression of HMGB1mRNA decreased gradually from20to48h afterGL+LPS stimulation.The expression of HMGB1mRNA in GL group wassignificantly lower than that in the LPS group at the same point of time(P<0.01).HMGB1protein in nucleus was abundant before stimulation,weobserved a reverse phenomenon in cytoplasm.HMGB1protein in cytoplasmincreased gradually from8to48h after LPS stimulation, HMGB1protein incytoplasm increased gradually from8to12h after GL+LPSstimulation(P<0.01).HMGB1protein in cytoplasm decreased graduallyfrom16to48h after GL+LPS stimulation(P>0.05).The cytoplasm HMGB1protein in GLgroup was significantly lower than that in the LPS group at thesame point of time(P<0.01).HMGB1protein in nucleus increased at8h afterLPS and GL+LPS stimulation,decreased gradually from12to 48h(P<0.01).The nucleus HMGB1protein in GL group was significantlylower than that in the LPS group at the same point of time(P<0.01).We observed strong green fluorescence of HMGB1in nucleus,andweak fluorescence in cytoplasm.The fluorescence of HMGB1decreased at8h after LPS stimulation,gradually enhanced in both nucleus and cytoplasmfrom12to24h.The fluorescence of HMGB1gradually enhanced incytoplasm from8to12h after GL+LPS stimulation,decreased at24h.TheHMGB1fluorescence in GL group was significantly lower than that in theLPS group at the same point of time.The level of HMGB1in the cell culture supernatant gradually increasedfrom8to48h after LPS and GL+LPS stimulation(P<0.01).The level ofHMGB1in supernatant in GL group significantly lower than that in the LPSgroup at the same point of time(P<0.01).Conclusions：1.GLsignificantly inhibited the transposition and release of HMGB1inmacrophages which induced by LPS.GL decreased the level of HMGB1inmacrophages cell culture supernatant.2.GL suppressed the expression of HMGB1mRNA in macrophagesinduced by LPS.GL significantly inhibited the synthesis of HMGB1inmacrophages which had been induced by LPS. Objective：We use LPS and GL+LPS to stimulate RAW264.7cells respectivelyand detect NF-κB and AP-1signaling pathway related moleculars,seeking toelucidate the molecular mechanism of glycyrrhizin inhibitelipopolysaccharide induced HMGB1releasing and expression fromRAW264.7cellsMethods：The RAW264.7cells was stimulated with100μg/L LPS and100μg/LLPS+1mmol/L GL respectively,culture cells for0,4,8,12,16,20,24and48h.The cell culture supernatant was collected,in which TNF-α and IL-6wasdetermined by ELISA.The expression of TLR4/MD2receptor was detectedby FCM.The contents of MyD88,phospho-TAK1,phospho-p38MAPK andNF-κB p65protein were determined by western blot.The contents andlocalization of NF-κB and AP-1in cells were observed byImmunofluorescence/Laser confocal microscopy.Results：The excessive expression of TLR4/MD2receptor on cells was detectedbefore stimulation.It decreased from4to8h and increased gradually from12 to48h after LPS stimulation(P<0.01).Similarly phenomenon was observedafter GL+LPS stimulation.The TLR4/MD2receptor expression in GLgroupwas significantly lower than that in the LPS group at the same timepoint(P<0.01).The level of MyD88,phospho-TAK1and NF-κB p65protein was lowbefore stimulation,it increased from4to48h after LPS stimulation(P<0.01).The level of MyD88,phospho-TAK1and NF-κB p65protein was increasedfrom12to48h after GL+LPS stimulation(P<0.01).The level ofMyD88,phospho-TAK1and NF-κB p65protein in GL group wassignificantly lower than that in the LPS group at the same point of time(P<0.01).The green fluorescence of NF-κB was in cytoplasm mostly beforestimulation,it enhanced both in cytoplasm and nucleus from4to24h afterLPS stimulation.The fluorescence of NF-κB in cytoplasm enhanced from4to24h after GL+LPS stimulation,but we didn’t observed the fluorescence ofNF-κB in nucleus.The fluorescence of NF-κB in cytoplasm in GLgroup wassignificantly lower than that in the LPS group at the same time points.The expression of phospho-p38MAPK protein increased from8to48hafter LPS stimulation(P<0.01).The expression of phospho-p38MAPKprotein keep unchanged from0to48h after GL+LPS stimulation(P>0.05).The expression of phospho-p38MAPK protein in GL group wassignificantly lower than that in the LPS group at the same point oftime(P<0.01).The green fluorescence of AP-1was in nucleus mostly before stimulation,it gradually enhanced from8h to24h after LPS and GL+LPSstimulation,which in GL group was significantly lower than that in the LPSgroup at the same time point.The level of TNF-α and IL-6in cell culture supernatant was graduallyincreased from4to48h after LPS and GL+LPS stimulation(P<0.01).Thelevel of TNF-α and IL-6in cell supernatant in GL group was significantlylower than that in the LPS group at the same point of time(P<0.01).Conclusions：1.LPS induced the expression and release of HMGB1through theTLR4/MD2â†'MyD88â†'TAK1â†'p38MAPKâ†'AP-1signaling pathway.GLinhibited the expression and release of HMGB1through blocking thesignaling pathway mentioned above.2.GL inhibited the release of TNF-α and IL-6through blocking theTLR4/MD2-NF-κB signaling pathway.GL inhibited the massive release ofTNF-α and IL-6through suppressing the expression and release of HMGB1at advanced stage.