ABSTRACT
Objective To investigate the role of anaphylatoxin C3a on epithelial mesenchymal transition(MTT) in normal human bronchial epithelium cells and its molecular mechanism.Methods Normal human bronchial epithelium cells BEAS-2B were cultured,and divided into control group,rhC3a stimulation group,rhC3a+ C3a receptor(C3aRA)antagonist group,the morphological changes of cells were observed by microscope;cell proliferation was detected by MTT;the expression of TGF-β1 protein level in cell supernatant was evaluated by ELISA;the expression of C3aR mRNA and EMT related indicators mRNA changes were detected by RT-PCR;the expression of C3aR and Smad2/3,p38 MAPK pathway proteins were detected by Western blot.Results Cell morphology in 30,50 nmol/L rhC3a stimulation group was changed from normal cobblestone like to spindle shape,cell morphology in C3aRA antagonist group had no significant change when compared with the control group.The cell proliferation was reduced in 50 nmol/L rhC3a stimulation group(P=0.047);the levels of TGF-β1,p-Smad2/3,p-p38-MAPK protein were increased(P＜0.05),C3aR mRNA and protein levels were also significantly increased (P＜0.05) in 30 nmol/L rhC3a stimulation group when compared with control group,but the addition of 1 μmol/L C3aRA could reduce their expressions(P＜0.05).30 nmol/L rhC3a could induce the up regulation of α-SMA and N-cadherin mRNA,and decrease the expression of E-cadherin mRNA,adding 1 μmol/L C3aRA can antagonize this process (P＜0.05).Conclusion Anaphylatoxin C3a can induce EMT in normal human bronchial epithelium cells by combining C3aR,its mechanism may be involved in activating Smad2/3 and p38-MAPK pathway.
ABSTRACT
Objective To explore PGD2 biological characteristics in L-929 mice lung fibroblasts cell by Laropiprant is a specific in-hibitor of PGD2 receptor regulation of TGF-β1/Smads signaling pathway .Provide further basis research to explore the molecular mechanisms of airway fibrosis .Methods The cells divided by Laropiprant concentration 0 .3 μmoL group ,1 .0 μmoL group ,3 .0μmoL group ,10 .0 μmoL group ,30 .0 μmoL and the control group .Each group were added TGF-β2 (2 .5 ng/mL) were cultured for 24 h after stimulation with the corresponding concentrations of Laropiprant 24 h ,TGF-β1 Smad3 and Smad4 expression were detected by PCR and WB ,respectively .A randomized approach ,using different concentrations of Laropiprant acts on cells at different times (12 ,24 ,48 ,72 h) by MTT assay for cell growth Laropiprant inhibition .Results TGF-β1 ,Smad3 and Smad4 mRNA expression de-creased with the addition of Laropiprant concentration increases ,there was significant difference compared with the control group with (P<0 .05) .TGF-β1 ,Smad3 and Smad4 protein expression decreased with the addition of Laropiprant concentration increases ,there was significant difference compared with the control group with (P< 0 .05) .Cell growth inhibition rate decreased with Laropiprant concentration increasing and cell culture time growth ,Laropiprant in concentration 1μml ,reaction time was 24 to 36 h ,the cell growth inhibition was significantly improved .Conclusion PGD2-DP1 expression in L-929 mouse lung fibroblasts cell may be associated with TGF-β1/Smads regulation .growth inhibition rate decreased with Laropiprant concentration increasing and cell culture time growth ,re-action time prolong ,the cell growth inhibition was significantly improved .