The effects of β-casomorphin 7 on mucus production

Abstract

Background: The common belief that milk consumption increases respiratory mucus remains purely anecdotal and lacks appropriately controlled studies. Beta-casomorphin 7 (β-CM7), an opioid peptide released in the digestion of bovine milk, has been shown to increase mucus production in perfused rat intestine, and in human intestinal epithelial cells in vitro, via a μ-opioid signalling mechanism. However, the direct effect of β-CM7 on mucus secretion in respiratory cells remains unknown. Aims: This study aimed to test the hypothesis that β-CM7 increases mucus production in human bronchial epithelial cells (HBECs) in vitro, and human mucosal explant cultures ex vivo, and to investigate whether any effect present is due to a μ-opioid signalling mechanism. Methods: Air-liquid interface cultures of differentiated human bronchial epithelial cells (N=3), and human nasal mucosal explant cultures (N=7) were treated with β-CM7 (1x10-4M) for 24 hours, after a two-hour pre-incubation in control medium, or medium with naloxone, an opioid inhibitor. An enzyme-linked lectin assay (ELLA) was used to measure secreted mucin glycoprotein in the culture medium, and changes in expression of mucin genes MUC5AC and MUC5B in cell cultures were determined using real-time qPCR. Mucin content was normalised to total DNA content (cells) as an estimate of cell number, or baseline mucin secretion (explants). Results: In ALI cultures of HBECs, treatment with β-CM7 increased mucin secretion to 179% ± 38% compared to untreated controls (p < 0.05) at 4 hours, which was inhibited by naloxone; in naloxone-inhibited samples mucus secretion following β-CM7 treatment was not significantly different from untreated, uninhibited controls. Increases in MUC5AC and MUC5B expression were also observed at 4 hours; however increases were not statistically significant. In contrast, mucin secretion did not change following BCM-7 treatment of tissue explants, likely due to wide morphological and pathological variability between samples noted on histological examination. Discussion: These data suggest for the first time, that in the human respiratory epithelium in vitro, β-CM7 stimulates a considerable increase in mucin expression and secretion, and that higherpowered studies and in vivo models are required for further validation. Several methodological and practical considerations relevant to future work are identified.