Study On The Release Of Inflammatory Cytokines In Co-culture Of Human Bronchial Epithelial Cells And Neutrophils And The Inhibition Of Puerarin

Objective 1, To Construct the co-culture system of human bronchial epithelial cells (BEAS-2B cells) with human neutrophills.2, To investigate the effect of puerarin on protein release and gene expression of the inflammatory cytokines in co-culture of BEAS-2B cells and human neutrophills.3, To investigate the effect of puerarin on the NF-κB and p38 MAPK signal transduction pathway.Methods 1, the co-culture system of human bronchial epithelial cells with human neutrophil were constructed by immunomagnetic beads method.2, There were 6 experimental groups:BEAS-2B cells cultured alone group, neutrophils cultured alone group, BEAS-2B co-cultured with neutrophils group, BEAS-2B cells and neutrophils co-cultured in presence of 50μg/ml puerarin group, BEAS-2B cells and neutrophils co-cultured in presence of 100μg/ml group, and BEAS-2B cells and neutrophils co-cultured in presence of 200μg/ml puerarin group.3, BEAS-2B cells and neutrophills were cultured respectively, co-cultured together, and co-culture with puerarin (50μg/ml, 100μg/ml,200μg/ml) for a pre-determined time. Cytokines in culture supernatant were evaluated by protein array after incubated 12 h. And IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 were quantified by enzyme linked immunosorbent assay(ELISA) after incubated 2 h,6 h,12 h and 18 h. The mRNA gene expression of IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 was evaluated by Real time quantitative polymerase chain reaction (Real-time qPCR) after incubated 12 h. The NF-κB and p38 MAPK Signal transduction pathway were evaluated by Western blot after incubated for 30 min.Results 1, A small amount of IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 were detected in supernatant of BEAS-2B cells cultured alone and neutrophils cultured alone group after incubated 2h,6h,12h, and 18h. IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 protein release in the culture supernatant was strikingly elevated when BEAS-2B cells were co-cultured with human neutrophils compared to that in supernatant of BEAS-2B cells cultured alone and neutrophils cultured alone. The expression of these cytokines was significantly decreased in the supernatant of BEAS-2B cells and neutrophils co-cultured with puerarin.2, The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 mRNA gene expression in BEAS-2B cells, but not neutrophils after 12 h incubation comparing with that in BEAS-2B cells and neutrophils cultured respectively. The expression of IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 mRNA gene was significantly up-regulated in BEAS-2B cells co-cultured with human neutrophils. The mRNA gene expression of these cytokines was significantly down-regulated in BEAS-2B cells co-cultured with human neutrophils by puerarin.3, The expression of Phospho-IκB and Phospho-p38 MAPK was significantly up-regulated in BEAS-2B cells co-cultured with human neutrophils. And the expression of these proteins was significantly down-regulated in BEAS-2B cells co-cultured with human neutrophils by puerarin.Conclusion 1, Human bronchial epithelial cells could be activated to express IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 mRNA gene and release IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TIMP-2 protein when co-cultured with human neutrophils.2, This effect could be inhibited by puerarin.3, The inhibition effect of puerarin was achieved possibly by inhibiting the NF-κB and p38 MAPK signal transduction pathway.