Glycyrrhizin Regulates Epithelial-mesenchymal Transition In Airway Epithelial Cells Through The Inhibition Of HMGB1/TGF-?1 Feedback Circuit

Objective:In recent years,the effect of epithelial mesenchymal transformation(EMT)on chronic airway diseases such as asthma and chronic obstructive pulmonary disease(COPD)has been deeply studied.As an important pathophysiological link of airway remodeling,EMT has promoted the progress and even malignant transformation of asthma and COPD.At present,there are no specific drugs and treatments that can effectively regulate EMT,thus it is of great significance to study how to delay and impede the EMT progress.The pathogenesis of EMT is complex and there are many influencing factors,among which the activation of transforming growth factor ?1(TGF-?1)and its downstream pathways play a significant role.Taking COPD as an example,TGF-?1 is significantly increased in serum and tissue samples of COPD patients,promoting the progress of its chronic inflammatory responses and airway remodeling.Studies have shown that high-mobility group boxl(HMGB1)is to some extent involved in the process of TGF-?1 induced EMT.As a natural inhibitor of HMGB1,glycyrrhizin can bind to HMGB1,reducing its protein activity and inhibiting the inflammatory responses caused by the increase of HMGB1 level.This study:intended to investigate the interaction between TGF-?1/smad2/3 pathway and HMGB1 expression in EMT progress,and to explore whether glycyrrhizin can suppress the development of TGF-?1 induced EMT,as well as its relevant mechanism.MethodsTGF-?1 was used to induce EMT changes in A549 and BEAS-2B cells.RNA interference and lentiviral infection experiments were conducted respectively,and the transfection or infection efficiency was detected by real-time fluorescent quantitative PCR experiment.To explore the effect of HMGB1 on TGF-?1-induced EMT,changes in relevant protein markers such as E-cadherin and vimentin were detected by western blot experiment.The concentration of HMGB1 in cell culture supernatant was tested by Elisa kit to investigate the translocation process of HMGB1 from intracellular to extracellular.CCK-8 kit was used to detect the survival rate of A549 or BEAS-2B cells treated with different concentrations of glycyrrhizin,in order to determine the appropriate glycyrrhizin treatment concentration.Epithelial cells were divided into normal control group,negative control group,HMGB1 overexpression group and glycyrrhizin+HMGB1 overexpression group.Western blot was used to investigate the effect of glycyrrhizin treatment on EMT caused by elevated HMGB1 level.The two epithelial cells were then respectively divided into normal control group,TGF-?1 treatment group.glycyrrhizin+TGF-?1 treatment group,western blot and immunofluorescence staining were conducted to evaluate the effect of glycyrrhizin on the EMT markers changes induced by TGF-?1,while the Transwell migration assay was used to test the migration ability of cells in different groups.Finally,changes in protein expression of TGF-?1/smad2/3 signaling pathway were detected by western blot.Results1.Knockdown of HMGB1 can reverse EMT changes induced by TGF-?1 in A549 and BEAS-2B cells.After treated with exogenous TGF-?1 for 24h,both the expression and the release of HMGB1 were increased in the normal cells.Corresponding EMT changes occurred,showing that the expression of E-cadherin decreased,while the expression of vimentin increased,and the morphology of epithelial cells was observed to be more mesenchymal.However,the above EMT changes were reversed in the HMGB1 knockdown+TGF-?1 treated cell group.2.Glycyrrhizin inhibited the EMT progress in lung epithelial cells caused by HMGB1 lentivirus over-expression.The decreased E-cadherin level and increased vimentin level can be detected in HMGB1-overexpressed cells.A549 cells were treated with glycyrrhizin at 50,100 and 200 ? M for 24h,and BEAS-2B cells were treated with glycyrrhizin at 25,50 and 100 ? M for 24h.In the HMGB1 overexpression+glycyrrhizin cells,E-cadherin was significantly increased and vimentin was correspondingly decreased.3.Glycyrrhizin suppresses TGF-?1-induced EMT in lung epithelial cells by inhibiting HMGB1.After glycyrrhizin pretreatment at different concentrations for 2h,TGF-?1 was added into the medium and the cells were continued to be cultured for 24h.The concentration of HMGB1 in culture supernatant detected by the Elisa kit was significantly increased after TGF-?1 treatment,while glycyrrhizin pretreatment decreased the HMGB1 level in culture supernatant.Western blot and immunofluorescence staining showed the higher level of E-cadherin and the lower level of vimentin in cells treated with glycyrrhizin+TGF-?1 compared with cells treated with TGF-?1 only.4.Glycyrrhizin decreased the migration of epithelial cells induced by TGF-?1.In the TGF-?1 treatment group,the number of migrated cells increased significantly compared with the normal group,while the number of migrated cells decreased significantly in the glycyrrhizin+ TGF-?1 treatment group,which was negatively correlated with the concentration of glycyrrhizin.5.Glycyrrhizin can partially block the activation of TGF-?1/smad2/3 signaling pathway by inhibiting HMGB1.In HMGB1 overexpressed cells,the levels of TGF-?1,phosphorylated Smad2,and phosphorylated Smad3 proteins were higher than in normal cells,indicating that the TGF-?1/Smad2/3 signaling pathway was activated.In HMGB1-overexpressed with glycyrrhizin treated cells,the expression of the above proteins was reduced.The downstream Smad2/3 signaling pathway was activated by exogenous TGF-?1 in epithelial cells,however glycyrrhizin can reduce the corresponding pathway protein levels.ConclusionHMGB1 promotes the EMT progress induced by TGF-?1 in human lung epithelial cells.By inhibiting HMGB1,glycyrrhizin effectively suppresses the EMT changes caused by HMGB1 overexpression,reverses the TGF-?1-induced EMT process,reduces the migration ability of lung epithelial cells,and partially block the activation of TGF-?1/smad2/3 signaling pathway.