Figure 6

Single-domain antibody A1 inhibits the production of MMP-9 and IL-8 stimulated by cyclophilin A through the ERK–NF-κB signaling pathway. (A) THP-1 cells were pretreated with different concentrations of single-domain antibody (sdAb) A1 or cyclosporine A (CsA) for 2 hours before cyclophilin A (CypA) stimulation. The expression of cytoplasmic IκBα, nuclear factor NF-κB-p65 at 2 hours, the level of ERK1/2 protein and phosphorylation of ERK1/2 at 30 minutes were analyzed by western blot, while β-actin of cytoplasm protein and histone of nuclear protein were used as internal control respectively. (B) THP-1 cells were pretreated with PD98059 for 2 hours before adding CypA. The level of IκBα and NF-κB-p65 protein at 2 hours and matrix metalloproteinase (MMP)-9 activity at 24 hours after CypA stimulation were determined by western blot and (C) gelatin zymography separately. (D), (E), (F) THP-1 cells were pretreated with 1-pyrrolidine carbodithioic acid, ammonium salt (PDTC) and N-tosyl-l-phenyl-alanylchloromethyl ketone (TPCK) (two inhibitors of NF-κB) for 2 hours before adding CypA. After 24 hours, the culture supernatants were collected and the activity of MMP-9 determined using gelatin zymogram (D) and the Ray Bio Tech (Guangzhou, China) MMP-9 activity immunoassay kit (E) separately. The level of interleukin (IL)-8 in the culture supernatants was also tested by the R&D Systems (Emeryville, CA, USA) human CXCL8/IL-8 immunoassay kit (F) according to the manufacturer’s instructions. (A) to (D) Data are representative results from three independent experiments. (E), (F) Bar graphs show the mean ± standard error of the mean for each group, with n = 3 wells per group. #P <0.05, ##P <0.01 versus control group; *P <0.05, **P <0.01 versus CypA group.