The Effects Of NLRP3 Inflammasome Inhibition On EMT Induced By Silica In 16HBE Cells

Epithelial-mesenchymal transition(EMT)refers to the transformation of epithelial cells into cells with stromal cell morphology and properties under certain factors.Inhalation of silica can induce EMT in lung bronchial and alveolar epithelial cells.NLRP3 inflammasome is a large cytosolic multiprotein complex,which can be activated by various stimuli,such as silica and can regulate the inflammation through the body.Previous studies have shown that chronic inflammation is closely related to EMT.However,few studies reported the effects of NLRP3 inflammasome on silica-induced EMT.Objectives:To explore the potential role and molecular mechanisms of NLRP3inflammasome in silica-induced EMT in 16HBE cells,and to investigate whether pirfenidone could alleviate silica-induced EMT through inhibiting the activation of NLRP3 inflammasome.Methods:Human bronchial epithelial cells(16HBE)were cultured in RPMI-1640medium containing 10%FBS.Two groups were arranged for the preliminary experiments:control group and silica group.Cells in silica group were incubated with RPMI-1640 medium containing 25,50,100,200?g/cm~2 silica for 24,48,72 h.Cell viability was confirmed with CCK-8 assay.Cell morphology changes were observed by microscope.The expressions of E-cadherin,?-SMA were detected by Western Blot and immunofluorescence staining.Appropriate dosage and time for silica stimulation was determined by the above results.For the following experiments,we adopted shNLRP3 lentiviral to downregulate NLRP3 protein expression,selective NLRP3 inflammasome inhibitor MCC950(0.01?0.1?1?10?100?M)to inhibit the inflammasome activation,caspase-1 inhibitor Z-YVAD-FMK(10?M)to inhibit the downstream effector molecule of inflammasome,thereby studing the effects of NLRP3 inflammasome inhibition on silica-induced EMT from the gene expression and functional level.ELISA was used to determine the levels of IL-1?and IL-18 in cell supernatant.Immunofluorescence staining was used to detect the expressions of E-cadherin and?-SMA.Western Blot was used to examine the expressions of E-cadherin,?-SMA,NLRP3,ASC,caspase-1 p10.The effects of pirfenidone(0.05,0.1,0.2,0.4 mg/ml)on silica-induced EMT and NLRP3inflammasome activation in 16HBE cells were studied.Besides,the effects of NLRP3 inflammasome inhibition on EMT-related signaling pathways were studied.Results:Silica inhibited the cell viability in a dose and time-dependent manner.After silica treatment,16HBE cells underwent morphological changes,turned into spindled-like mesenchymal phenotype.Western Blot and immunofluorescence showed that 50?g/cm~2 silica stimulation for 72 h decreased the expression of E-cadherin,increased the expression of?-SMA.Meanwhile,the expressions of NLRP3 inflammasome associated proteins(NLRP3,ASC and caspase-1 p10)were upregulated.The secretive levels of IL-1?and IL-18 in cell supernatant increased continuouly after 50?g/cm~2 silica challenge for 3,6,12,24,48,72 h.Downregulating the expression of NLRP3 by shNLRP3,inhibiting the activation of NLRP3 inflammasome by MCC950,or suppressing the activation of caspase-1(effector molecule of NLRP3 inflammasome)by Z-YVAD-FMK,could alleviate silica-induced cell morphology changes and protein expression changes of E-cadherin,?-SMA.Western Blot results showed that silica significantly induced the phosphorylation of TAK1,P38 MAPK,ERK1/2,JNK,NF-?B,Smad2,GSK3?and the upregulation of Snail.MCC950(100?M)pretreatment reversed the phosphorylation of TAK1,P38 MAPK and the increase of Snail,while greatly inhibited the phosphorylation of ERK1/2,JNK,NF-?B,but showed no influence on Smad2 and GSK-3?.Moreover,pirfenidone showed no effects on cell viability,but effectively suppressed silica-induced NLRP3 inflammasome activation,IL-1?,IL-18secretion as well as the expression changes of E-cadherin,?-SMA.Conclusions:Silica challenge induced EMT accompanied with continuous activation of NLRP3 inflammasome in 16HBE cells.Suppressing the activation of NLRP3inflammasome at different steps(such as downregulating the expression of NLRP3,selectively inhibiting the activation of NLRP3 inflammasome or suppressing the effector molecule of NLRP3 inflammasom)could alleviate silica-induced EMT.Molecular biological researches suggest that NLRP3 inflammasome may regulate silica-induced EMT via IL-1?-TAK1-MAPK-Snail/NF-?B pathway.Moreover,pirfenidone,a clinically available drug approved for treating IPF,could inhibit silica-induced EMT through inhibiting the activation of NLRP3 inflammasome.