Effect Of Glycyrrhizic Acid Ammonium Salt On Corticosteroid Resistance Of Mononuclear Cells Exposed To Cigarette Smoke Extract

Objective To explore the effect of glycyrrhizic acid ammonium salt (GA)on corticosteroid resistance of human THP-1cells that was stimulated bycigarette smoke extact (CSE) and its mechanism.Methods THP-1cells were cultured in vitro as a reseach objective for theexperiment. An appropriate concentration of CSE（volume fraction of5%，10%)and100μmol/L、200μmol/L、400μmol/L GA at different time points（24h，48h，72h） were confirmed by methl thiazolyldiphenyl-tetrazolium bromide(MTT) method.(1) The cells were divided into control group, CSE group,CSE+GA group to detect the expression of Interleukin8(IL-8) levels insupernatants and histone deacetylase2(HDAC2). CSE+GA group treated withCSE overnight was pretreated with GA for1hour.After that, the cells weretreated with different concentrations of dexamethasone(10-5-10-11mol/L) for1hour and then stimulated with tumour necrosis factor α（TNF-α）overnight.CSE group was treated the same as GA group except GA intervention. Controlgroup was cultured without CSE and GA, given only the dexamethasone andTNF-α. Interleukin8(IL-8) levels in supernatants was measured by enzyme-linked immunosorbent assay (ELSA)(2)The cells were divided intoblank control group, scrambled oligonucleotide (SC) group, HDAC2-siRNAgroup, HDAC2-siRNA+GA group in order to study the mechanism ofglucocorticoid resistance. HDAC2-siRNA+GA group was given short silenceHDAC2-siRNA sequences plasmid gene then transfected into the cells byliposome.48hours later, the cells were treated with GA overnight.HDAC2-siRNA group was the same as HDAC2-siRNA+GA group except forGA intervention. SC group was given an empty plasmid withoutHDAC2-siRNA, oher interventions were the same as HDAC2-siRNA group.Blank group was given no intervention. real-time quantitative polymerase chainreaction (qRT-PCR) was used to analysis HDAC2mRNA expression. Westernblotting was used to analyse the expression of HDAC2,gluocorticoid receptorα（GR-α）, nuclear factor κB(NF-κB),(P65) and then analyze the correlationbetween this factors. The data were analyzed using SPSS16.0statisticalsoftware.Results (1) There was no significant effect on the grouth of THP-1cellswhen the cells being treated with concentration of5%,10%(volume fraction)CSE and concentration of100μmol/L,200μmol/L,400μmol/L GA;(2) Theinhibition rate of IL-8in the CSE+GA group was higher than CSE group, butlower than control group(P＜0.05);(3) The half-maximal inhibitoryconcentration of dexamethasone(IC50-Dex) in the CSE+GA group was lowerthan CSE group,but higher than control group(P＜0.05);(4) The expression ofHDAC2mRNA and protein in the HDAC2-siRNA group was lower than controlgroup and SC group, the expression levels were increased significantly aftercombining with GA(P＜0.05);there were no significant difference betweencontrol group and SC group(P＞0.05);(5) The expression of GR-αin CSE group was lower than control group,but the expression of NF-κB in CSEgroup was higher than control group,after treating with GA, the expression ofGR-αincreased and the NF-κB decreased in protein levels(P＜0.05);（6）Theexpression level of GR-α and NF-κ B （P65） protein were negativelycorrelated(r=-0.697,P＜0.01), the HDAC2and NF-κB （P65）protein werenegatively correlated(r=-0.667,P＜0.01),there is a positive correlation betweenGR-αand HDAC2in protein levels(r=0.646,P＜0.01).Conclusion (1) The THP-1cells which being cultured and stimulated withCSE were significantly reduced the sensitivity to dexamethasone, it indicatedthe existence of corticosteroid resisitance.(2) GA may reverse the glucocorticoidinsensitivity by up-regulating the expression of GR-α,HDAC2protein,anddowing-regulating the expression of NF-κB.