Triptophenolide reversing the process of epithelial-mesenchymal transition of pulmonary tracheal epithelial cells through NF-κB pathway
Objective To investigate the effect of triptophenolide on the process of epithelial-mesenchymal transition(EMT)in pulmonary tracheal epithelial cells.Methods Firstly,the cytotoxic effect of triptophenolide on Beas-2b cells was assessed using the cell counting kit-8(CCK-8)assay to determine the appropriate dosage of triptophenolide.The cells were divided into control group,model group,1 μmol/L-dose triptophenolide group,and 10 μmol/L-dose triptophenolide group.The control group was not subjected to any treatment,while cells in the model group were treated with 10 ng/mL transforming growth factor(TGF)-β1 combined with 10 ng/mL TNF-α for 48 h.In addition to the treatment in the model group,cells in the triptophenolide group were co-cultured with a final concentration of 1 μmol/L or 10 μmol/L triptophenolide for 48 h.Cell morphology changes were observed under a phase-contrast microscope.Immunofluorescence assay was performed to detect the expressions of EMT markers,including E-cadherin,αlpha smooth muscle actin(α-SMA),and Vimentin.RT-PCR and Western blot were used to quantitatively detect E-cadherin,α-SMA,and Vimentin mRNA and protein expressions.Additionally,the effect of triptophenolide on the NF-κB pathway and the expression levels of EMT-related transcription factors,including Snail family zinc finger 1(SNAI1),zinc finger E-box binding homeobox 1(ZEB1),and twist family BHLH transcription factor 1(TWIST1),were confirmed by Western blot.Results The treatment with 1 μmol/L and 10 μmol/L tripto-phenolide both increased cell viability and decreased the migratory capacity of cells in the model group.Immunofluorescence and Western blot results showed that triptophenolide inhibited the EMT induced by TGF-β plus TNF-α.Compared to the model group,1 μmol/L and 10 μmol/L triptophenolide both downregulated the fluorescence intensity of α-SMA and Vimentin proteins,while upregulated that of E-cadherin protein.Furthermore,the detection of EMT-related transcription factors showed that the protein expression levels of p-p65,TWIST1,ZEB1,and SNAI1 were all upregulated in the model group,whereas triptophenolide downregulated the above EMT-related transcription factors.Conclusion Tripto-phenolide reduces the expressions of EMT-related transcription factors of SNAI1,ZEB1,and TWIST1 by inhibiting NF-κB activity,thereby inhibiting EMT in Beas-2b cells that is induced by TGF-β plus TNF-α.
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维普
