Mechanism Of Glycyrrhiza In Anti-inflammatory Effects By Inhibiting The TLR4 Signaling Pathway

BackgroundInflammation refers to the defense response of the living tissue with the ascular system to the injury factor,characterized by a variety of inflammatory cells and inflammatory cytokines involved in complex process,which is base with varieties of diseases,such as respiratory disease,cardiovascular disease,metabolic disease,inflammatory bowel disease,etc.,involving multiple organizations organs lesions,the common characteristic of these diseases characterized by abnormal body inflammatory reaction control and immunization of imbalances.Glycyrrhiza is a traditional Chinese medicine in our country,Licochalcone A is its main chemical composition.In recent years,more and more studies have found that the active ingredients of Glycyrrhiza extract have better anti-inflammatory effects,which can play a significant inhibitory role in the expression pathways of inflammatory factors such as IL-6 and TNF-α.Glycyrrhiza extract showed obvious anti-inflammatory activity in various animal models,such as sodium sulfate-induced acute colitis model in mice and atherosclerosis model in mice.Toll like receptor(TLR)is an important pathogen pattern-recognition receptor,which plays a critical role in activating innate immunity and inflammatory response.The abnormal expression or regulation of key proteins in the signaling pathway can lead to immune disorders and paradoxical inflammatory reaction.More and more studies have confirmed that toll like receptor 4(TLR4)is a transmembrane protein.After activation,NF-k B expression in the downstream nuclear transcription factor is activated,resulting in the release of a large number of inflammatory factors(such as IL-6,TNF-α,IL-1β,etc.).Based on the previous reports,it is worthwhile to investigate the effects of Glycyrrhiza plays an anti-inflammatory role by inhibiting TLR4.To address the above issues,we used the extract of Glycyrrhiza and Licochalcone A as the in vivo and vitro model investigate whether through inhibiting TLR4 to play an anti-inflammatory.ELISA,qRT-PCR,Western blot,molecular docking,molecular dynamics and other technologies were used to examine the interactions between Glycyrrhiza and TLR4.MethodsPart I: the effects of LicoA and Glycyrrhiza extract on inflammatory mice and its mechanism:1.Inflammatory mice model was established,and100 male C57BL6 mice wererandomly divided into control group,model group intraperitoneal injection of LPS(10mg/ml),low dose group(2.5g/kg),medium dose group(5g/kg),high dose group(10g/kg)dose group of Glycyrrhiza extract,LicoA low dose group(25mg/kg),Glycyrrhiza extract medium dose group(50mg/kg)and Glycyrrhiza extract high dose group(100mg/kg).LicoA and Glycyrrhiza extract were intragastrically administered once daily for seven days.2.After establishing the model 24 h,The levels of Tumour necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in bronchoalveolar lavage fluid(BALF),serum and lung tissues in LPS-induced rats were detected.Furthermore,we detected the pulmonary protein expressions of MAPK and NF-κB pathway-associated proteins which downstream of TLR4 pathway.Immunohistochemical analysis was used to observe the pathological changes of lung tissue.Part II: The effects of LicoA and Glycyrrhiza extract on the differentiation of THP-1 macrophages.1.The lipopolysaccharide(LPS)-induced human acute mononuclear leukemia cell THP-1which were differentiated to macrophages by Phorbol myristate acetate(PMA)-treated,were used to construct an in vitro inflammatory cell model.2.THP-1 inflammatory cell model was established.After 24 h treatment with different concentrations of LicoA(5,10,20μM)and Glycyrrhiza extract(100,200,400μg/ml).The release amount of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)in cell culture supernatants was detected by ELISA;the expression level of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)mRNA was detected by qRT-PCR.3.THP-1 inflammatory cell model was established.The formation of LPS/TLR4 complex was detected by immunofluorescence,and the expression of TLR4 pathway-associated protein was detected by Western blotting.4.THP-1 inflammatory cell model was established.THP-1 cells were pretreated by adenovirus transfected with TLR4 for 48 h.The release amount of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)in cell culture supernatants was detected by ELISA;the expression level of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)mRNA was detected by qRT-PCR;The expression of TLR4 pathway-associated protein was detected by Western blotting.5.The relationship between Glycyrrhiza and TLR4 was observed by molecular docking and molecular dynamics techniques.Results1.After intraperitoneal injection of LPS in rats,can successfully construct an in vivo inflammatory model.bronchoalveolar lavage fluid(BALF)and serum in LPS group compared with the control group,pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)expression was significantly increased.2.Different concentrations of LicoA and Glycyrrhiza extract,bronchoalveolar lavage fluid(BALF)and serum’s expression level of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)mRNA was s dose-dependently reduced by qRT-PCR,its anti-inflammatory effect was the same as ELISA assay.In addition,the expression of TLR4 pathway-associated protein was significantly reduced following LicoA and Glycyrrhiza extract administration in a dose-dependent way(P< 0.05).3.HE staining observed the lung tissue of mice.The model group showed obvious alveolar space widening,alveolar shapechange,inflammatory cell infiltration and so on.4.LPS-induced THP-1 cells can successfully construct an in vitro inflammatory cell model,LPS group compared with the control group,pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)expression was significantly increased.(P<0.05).5.Different concentrations of LicoA and Glycyrrhiza extract,the expression level of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β)mRNA was s dose-dependently reduced by qRT-PCR,its anti-inflammatory effect was the same as ELISA assay(P<0.05).6.Immunofluorescence showed that different concentrations of LicoA and Glycyrrhiza extract could inhibit the binding of LPS to THP-1 cell membrane.In addition,the fluorescence intensity of extracellular lipopolysaccharide decreased in a dose-dependent way.In addition,the expression of TLR4 pathway-associated protein was significantly reduced following LicoA and Glycyrrhiza extract administration in a dose-dependent way.(P<0.05).7.The results of qRT-PCR and Western blot showed that overexpression of TLR4 can partially block the anti-inflammatory effect of LicoA and Glycyrrhiza extract on THP-1 cells.8.Molecular docking showed that there was a strong binding ability between TLR4 and Glycyrrhiza,which indicated that Glycyrrhiza could interact directly with TLR4.And the molecular dynamics results showed that Glycyrrhiza could stably bind to TLR4.ConclusionGlycyrrhiza regulates LPS-induced inflammation by inhibiting the signaling pathway of TLR4.