ABSTRACT
Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.