Abstract:

Objective To investigate the effects of hypertonicity on the secretion of Mucin (MUC) 5AC of 16HBE cells and possible mediate effects of Wnt/β-catenin pathway. Methods The hypertonic medium was prepared by the solution of high concentration of sodium chloride and the16HBE cells were stimulated by the hypertonic medium. The 16HBE cells were divided into the negative control group cultured in normal medium and groups stimulated by the hypertonic medium for 3, 6, 9, and 12 h. The secretion volume of MUC5AC was detected by the ELISA. The expressions of Wnt family proteins of human airway, i.e. Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt10b, were detected by the Western blotting. The 16HBE cells were transfected by the specific interference RNA (siRNA) of Wnt4 and stimulated by the hypertonic medium. The secretion volumes of MUC5AC in culture supernatants were detected by the ELISA. The expressions of signaling molecules related to classic Wnt/β-catenin pathway were detected by the Western blotting. The translocation of nuclear factor (NF)-κB p65 was visualized by the cell immunofluorescence. Results The secretion volumes of MUC5AC in supernatants and cytoplasm of 16HBE cells cultured by the hypertonic medium were significantly higher than those of the control group. The secretion volumes of MUC5AC exhibited a time-dependent manner in a certain range of concentration (P<0.05). Compared to the negative control group, the expression levels of Wnt1, Wnt2, Wnt3a, Wnt5a, Wnt7a, Wnt10a, and Wnt10b of the hyperosmotic groups were not significantly different (P>0.05), while the expression levels of Wnt4 significantly increased (P<0.05). The relative expression levels of β-catenin and Cyclin D1 in cells of the hyperosmotic groups were significantly higher than those of the negative control group. The cell immunofluorescence indicated that nuclear translocation of NF-κB p65 occurred (P<0.05). The secretion volumes of MUC5AC in supernatants and cytoplasm of the hyperosmotic groups that were transfected by the Wnt siRNA were significantly lower than those of the hyperosmotic groups that were not transfected by the Wnt siRNA (P<0.05). The levels of β-catenin and CyclinD1 and the translocation of NF-κB p65 in cells were also significantly inhibited (P<0.05). Conclusion The Wnt/β-catenin signaling pathway plays an essential role in the hypersecretion of MUC5AC in 16HBE cells induced by the hypertonicity.

Key words: osmotic pressure, mucin5AC, Wnt/β-catenin signaling pathway, nuclear factor-κB