The Molecular Mechanism Of Bitter Receptor Agonists Ameliorating LPS-induced Inflammatory Responses In Airway Epithelial Cells

Background: Bitter taste receptors(TAS2R)are belonging to the G protein coupled receptor(GPCR).It has about 25 functional genes encoding bitter taste receptor proteins,which are found in the oral and throat,as well as endocrine,circulatory,digestive and respiratory systems.The expression of TAS2 R receptors in human airway epithelium has been demonstrated.Additionally,picrin activation of bitter receptors improves lipopolysaccharide(LPS)-induced inflammatory responses by inhibiting the NF-κB signaling cascade,suggesting that TAS2 R has a significant anti-inflammatory potential.However,the regulatory effect and mechanism of TAS2 R on the physiological function of airway epithelial cells have not been fully understood.Purpose: To explore the expression and localization of TAS2R10/14 in human bronchial epithelial cells,and observe the inflammatory response mechanism of human bronchial epithelial cells induced by LPS,and the effect of inflammatory response on the expression of TAS2R10/14,MUC5 AC and NF-κB.To explore the mechanism of TAS2R10/14 activation by picrin to improve the inflammatory response of human bronchial epithelial cells induced by LPS.Methods: Human lung tissues were collected from surgical subjects(no tumor cells confirmed by immunohistochemistry),including 8 subjects with a history of chronic obstructive pulmonary disease(COPD)or asthma and 10 subjects of no history of COPD or asthma,to be the control group.The expressions of TAS2R10 and TAS2R10 proteins in human bronchopulmonary tissue were observed by immunohistochemistry.16 HBE human airway epithelial cells were cultured And then stimulated with 1μg/ml LPS and100μg/ml LPS,IL-6,TNF-α and MUC5 A protein in the supernatant were assayed by using ELISA,the expression of TAS2R10/14 were detected with immunofluorescence.Cells were further divided into the following groups: normal control group,100μg/ml LPS group,100μg/ml LPS plus 100μmol/l limonin group,100μg/ml LPS plus100μmol/l naringin group,and 100μg/ml LPS plus quinine 250μmol/l group.IL-6,TNF-α and MUC5 A protein in the supernatant were also detected by ELISA kit.The expressions of TAS2R10/14,and MUC5 AC were observed by cell immunofluorescence and confocal laser microscope.Nuclear transport activation of NF-κB were detected by NF-κB activation,Nuclear Translocation Assay Kit.Results:The expression of TAS2R10 and TAS2R14 in airway epithelial cells and smooth muscle cells could be observed by immunohistochemical staining,and their expression in the chronic inflammation group appears to be lower,compared to the non-chronic inflammation group.Immunofluorescence staining also observed the expression of TAS2R10 and TAS2R14 in cultured 16 HBE cells in vitro.Compared with normal control group,IL-6,TNF-α and MUC5 AC protein in airway epithelial cells were significantly increased in LPS group(P < 0.05),while the concentrations of IL-6,TNF-α and MUC5 AC protein in airway epithelial cells were significantly decreased after bitter taste receptor agonists limonin,naringin and quinine pretreatment(P < 0.05).Immunofluorescence revealed TAS2R10 and TAS2R14 were mainly expressed in cell membrane and cytoplasm.After LPS stimulated for 24 h,the expression of TAS2R10 and TAS2R14 were decreased,the expression of MUC5 AC was significantly enhanced,and the expression of NF-κB showed a significant nuclear transport activation tendency,compared with the normal group,P < 0.05.After bitter receptor agonists pretreatment,the fluorescence expression of TAS2R10 and TAS2R14 was increased,and the expression of MUC5 AC and NF-κB nuclear transport were also decrease,compared with LPS group,P < 0.05.Conclusions: In this study,we confirmed the expression of TAS2R10 and TAS2R14 in 16 HBE epithelial cells.Bitter taste receptor agonists mitigate LPS-induced cellular inflammatory cytokine secretion by inhibiting NF-κB signaling cascades in 16 HBE epithelial cells.