Effects And Mechanisms Of Shensu Drink On β-defensin-2 In Human Bronchial Epithelial Cells

ObjectiveExplore the correlation between the influence of Shensu Drink on the expression of β-defensin-2 in human bronchial epithelial cells and TLR4-NFκB signaling pathway.Methods1.Collect cardiac blood and produce drug-containing serum after using the method of intragastric administration to give Shensu drink and its parts liquid medicine to male SD rats.2.Subculture 16 human bronchial epithelial cells (16HBE).3.In order to build 16HBE inflammation model, the proliferation activity of 16HBE and the content of TNF-a, IL-8 in culture supernatant was determined at different time points (3h,6h,12h,24h) after LPS (1mg/L) stimulation.4.After administration of drug-containing serum,the content of hBD-2 in 16HBE was determined using western blotting at different time points (3h,8h).5.After administration of drug-containing serum,the content of TLR4mRNA,NFKBp65mRNA and hBD-2mRNA in 16HBE was determined using RT-PCR at different time points (0.5h, 1h,3h,5h,8h).6.After administration of drug-containing serum,the activity of NFκB in 16HBE was determined using filter plate assay at different time points (0.5h,1h,3h,5h,8h).Results1.Successfully cultured 16 human bronchial epithelial cells (16HBE). 16HBE were polygonal, irregular and cobblestone-like monolayer growth.Generally, passaging one generation needed 3 to 5 days. After passaging for 3-4h, the cells started to adhere to bottom of the culture bottle, then, appeared split phase. The cells splitted to be clusters-like,network-like and finally connected into pieces.2.Successfully builded 16HBE inflammation model. The proliferation activity of 16HBE and the content of TNF-a, IL-8 in culture supernatant significantly increased compared with those of blank group (p<0.05).3.The influence of Shensu Drink on the expression of β-defensin-2 protein in 16HBE. The content of β-defensin-2 protein in the group of full formula, group of nourishing qi to relive superficies and group of relieving cough and resolving phlegm significantly increased at 3h,8h compared with that of model group (p<0.05).4.The influence of Shensu Drink on the expression of hBD-2mRNA, NFκBp65mRNA and TLR4mRNA in 16HBE. The content of hBD-2mRNA in the group of full formula and group of nourishing qi to relive superficies significantly increased at lh,3h,5h compared with that of model group (p<0.05) and peaked at 3h. The content of hBD-2mRNA in the group of relieving cough and resolving phlegm significantly increased at 1h,5h compared with that of model group (p<0.05). The content of NFκBp65mRNA in the group of full formula, group of nourishing qi to relive superficies and group of relieving cough and resolving phlegm significantly increased at 3h,5h compared with that of model group (p<0.05). The content of TLR4mRNA in the group of full formula and group of nourishing qi to relive superficies significantly increased at 3h,5h,8h compared with that of model group (p<0.05). The content of TLR4mRNA in the group of relieving cough and resolving phlegm significantly increased at 5h,8h compared with that of model group (p<0.05).S.The influence of Shensu Drink on the activity of NFκB in 16HBE. The activity of NFκB in the group of full formula, group of nourishing qi to relive superficies and group of relieving cough and resolving phlegm significantly increased at lh,3h,5h,8h compared with that of model group (p<0.05).ConclusionsShensu Drink probably effects on TLR4-NFκB signaling pathway to promoting the expression of hBD-2 in human bronchial epithelial cells. HBD-2 is likely to be the target spot in the process of Shensu Drink exerting its efficacy.