Objective: To explore the expression of 4-hydroxy-2-nonenal (4-HNE) in human bronchial epithelial cells (16-HBE) caused by cigarette smoke condensate (CSC), and to study the effects of ginkgolide B on 4-HNE expression and the role of 4-HNE on neutrophils chemotaxis.
Methods: Immunohistochemical and Western blot method were used to estimate the expression of 4-HNE-modified protein in 16-HBE of different groups (control group, 1 microg/ml or 10 microg/ml CSC stimulation for 4 h, and 10 microg/ml CSC stimulation for 1, 4, 8, 12, 24 or 30 h). 16-HBE were incubated with 100 micromol/L ginkgolide B for 24 h before CSC (10 microg/ml) stimulation, and the expression of 4-HNE-modified protein were recorded at 1, 4, 8 and 12 h after CSC stimulation. The chemotaxis of neutrophils was measured in different concentrations of 4-HNE (0.1, 1 and 10 micromol/L).
Results: (1) The expression of 4-HNE-modified protein was elevated significantly in CSC stimulating groups (immunohistochemistry: 2.12 +/- 0.38, 2.69 +/- 0.42, Western blot: 100.2 +/- 6.3, 72.3 +/- 6.1) compared with the group without CSC stimulation (immunohistochemistry: 1.25 +/- 0.37, Western blot: 122.4 +/- 4.2, all P < 0.01). (2) The expression of 4-HNE-modified protein in groups treated with 10 microg/ml CSC in different times (1, 4, 8, 12, 24, 30 h, immunochemistry: 2.67 +/- 0.46, 2.69 +/- 0.42, 2.71 +/- 0.48, 2.72 +/- 0.56, 2.93 +/- 0.11, 2.92 +/- 0.20, Western blot: 73.2 +/- 8.3, 72.3 +/- 6.4, 72.6 +/- 9.2, 71.5 +/- 8.1, 54.4 +/- 3.6, 56.7 +/- 4.4) were higher than the control group (immunochemistry: 1.25 +/- 0.35, Western blot: 122.4 +/- 4.1, all P < 0.01). The expression of 4-HNE-modified protein in the 10 microg/ml CSC for 24 and 30 h was significantly higher than other groups. (3) There was a lower expression level of 4-HNE-modified protein in the groups with ginkgolide B (50, 100 micromol/L) pre-incubated for 24 h before 10 microg/ml CSC stimulation for 4 h (Western blot: 84.6 +/- 4.4, 101.2 +/- 4.4) than those without pre-incubated with ginkgolide B (Western blot: 72.5 +/- 6.4); all elevated significantly compared to the group without stimulating with CSC (all P < 0.01). (4) 4-HNE (0.1, 1, 10 micromol/L) showed higher neutrophil chemotaxis activity (92 +/- 12, 104 +/- 16, 131 +/- 12) than the control group (72 +/- 12, all P < 0.01). The numbers of chemotaxis neutrophils in 10 micromol/L 4-HNE group were higher than those in the 0.1 micromol/L and 1 micromol/L 4-HNE groups.
Conclusion: 4-HNE might be involved in neutrophil chemotaxis caused by cigarette smoking in the lung, and ginkgolide B could inhibit the expression of 4-HNE caused by CSC.