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Updated: Jun 5, 2026
Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
Published on: June 14, 2016
[Effects of endogeny polypeptide elafin on MUC5AC overexpression in human airway epithelial cells]
Hong-Mei Yu1, Qi Li, Juliy M Perelman
1Department of Respiratory Medicine, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Objective:
to explore the effects of endogeny polypeptide elafin on mucin (MUC) 5AC overexpression.
Methods:
eukaryotic expression vector pEGFP-N1-Elafin was constructed. HBE16 cells were cultured and divided into 6 groups:a control group, a cigarette smoke extract (CSE) stimulated group, a CSE and Elafin transfected group, a CSE and pEGFP-N1 transfected group, a single elafin transfected group, and a single pEGFP-N1 transfected group. After 24 h, the protein levels of phosphorylation Jun N-terminal kinase (p-JNK), phosphorylation extracellular signal-regulated kinase (p-ERK), p-P38 and inhibitor of NF-κB (IκB)α were detected by Western blot. The transcription activities of activator protein-1(AP-1) and nuclear factor κB (NF-κB) were detected by luciferase reporter gene detection system. The levels of MUC5AC protein and mRNA were detected by ELISA and RT-PCR.
Results:
in CSE group, there was a significant increase of MUC5AC protein (0.71 ± 0.04) mg/L and mRNA expression (0.81 ± 0.04), with elevation of p-JNK production (0.55 ± 0.03) microg/mg, p-ERK production (0.64 ± 0.06) microg/mg, p-c-Jun production (0.60 ± 0.07) microg/mg, AP-1 activity (7.49 ± 0.31) and NF-κB activity (4.42 ± 0.22), all significantly higher than those in the control group (t = 4.50 - 14.28, P < 0.01), and IκBα protein production was (0.27 ± 0.03) mg/L, significantly lower than that in the control group (t = 6.82, P = 0.008). Transfected recombinant elafin reduced MUC5AC protein (0.71 ± 0.04) mg/L and mRNA level (0.81 ± 0.04), decreased p-JNK (0.38 ± 0.04) microg/mg and p-ERK (0.31 ± 0.04) µg/mg production, inhibited AP-1 activity (2.60 ± 0.19) and NF-κB activity (2.55 ± 0.21), but increased IκBα protein (0.54 ± 0.03) µg/mg, compared with single CSE-stimulated group (all P < 0.05). p-P38 showed no significant change after CSE stimulation or transfection of elafin.
Conclusion:
Endogeny polypeptide elafin may down-regulate MUC5AC overexpression, and this is through the inhibition of AP-1 and NF-κB activation via effects on mitogen-activated protein kinases and IκB pathway by Elafin.
