Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2 production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity

Abstract

Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE(2) in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE(2) upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE(2) levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE(2) levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE(2) upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE(2). Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI.

Fig. 1.. Aspirin inhibits the LPS-induced COX-2 mRNA and PGE₂ production in vitro. (A) The effect of LPS on COX-2 mRNA levels. p-AMs were treated with LPS (1 μg/ml) for indicated times. Then the total RNA was isolated and the levels of COX-2 mRNA were determined by RT-PCR. (C) The effect of aspirin on LPS-induced COX-2 mRNA expression. The cells were pretreated with aspirin (3 mM) for 30 min and then treated with LPS (1 μg/ml) for the indicated times. (B, D) The relative expression of COX-2 mRNA compared with control. (E) The effect of aspirin (3 mM) on LPS-stimulated (1 μg/ml) PGE₂ production. The cells were pretreated with aspirin (3 mM) for 30 min and then treated with LPS (1 μg/ml) for 12 h. The levels of PGE₂ in the supernatants were determined by competitive ELISA method. Data are expressed as mean ± SE from 3 separate experiments. *P ＜ 0.05, compared with control, ^#P ＜ 0.05, compared with aspirin group.

Fig. 2.. PKC and PTP were involved in the suppressive effect of aspirin on LPS-stimulated COX-2 and PGE₂ production. (A, B) The effect of LPS (1 μg/ml) on the activity of PKC and PTP at the indicated times, respectively. (C, D) The effect of aspirin (3 mM) on the LPS-enhanced PKC activity and reduced PTP activity, respectively. The effect of calphostin C and POV on the suppressive effect of aspirin on LPS-induced COX-2 mRNA (E) and PGE₂ (F) expression. p-AMs were pretreated with the PKC inhibitor calphostin C (0.5 μmol/L), the PTP inhibitor POV (20 μmol/L), aspirin (3 mM), or combination of aspirin and calphostin C or POV, and then stimulated with LPS (1 μg/ml) for 1 h or 12 h for COX-2 mRNA and PGE₂ determination respectively. The levels of COX-2 mRNA and PGE₂ were determined as described in the methods section. Each bar represents mean ± SE from 3 independent experiments. *P ＜ 0.05, **P ＜ 0.01, compared with control group, ^#P ＜ 0.05, ^##P ＜ 0.01 compared with LPS group, ^лP ＜ 0.05, ^ллP ＜ 0.01 compared with aspirin group.

Fig. 3.. Aspirin inhibited LPS-induced NF-κB activation. (A) LPS stimulated NF-κB activation. Cells were treated with LPS (1 μg/ml) for indicated times. Then the DNA binding activity of NF-κB was measured by EMSA. (B) Aspirin treatment inhibited LPS-stimulated NF-κB activation. Cells were pretreated with aspirin (3 mM) for 30 min and then treated with LPS (1 μg/ml) for 60 min. Then the DNA binding activity of NF-κB was measured by EMSA. (C) The effect of LPS treatment on the level of IκBα at the indicated times. Cells were treated with LPS (1 μg/ml) for indicated times. Then the levels of IκBα were determined by western blot. (D) Aspirin treatment inhibited LPS-induced degradation of IκBα. Cells were pretreated with aspirin (3 mM) for 30 min and then treated with LPS (1 μg/ml) for 1 h. Then the levels of IκBα were determined by western blot. Each bar represents mean ± SE from six independent experiments. *P ＜ 0.05, **P ＜ 0.01 compared with control, ^#P ＜ 0.05, ^##P ＜ 0.01 compared with aspirin group.

Fig. 4.. PKC and PTP were involved in the suppressive effect of aspirin on LPS-induced NF-κB activation. The effect of calphostin C and POV on the suppressive effect of aspirin on LPS-induced NF-κB activation (A) and IκBα degradation (B). The p-AM cells were pretreated with calphostin C (0.5 μmol/L), POV (20 μmol/L), aspirin (3 mM), or combination of aspirin and calphostin C or POV for 30 min and then treated with LPS (1 μg/ml) for 1 h or 10 min for NF-κB and IκBα determination respectively. Then the activity of NF-κB and the level of IκBα were determined by EMSA and western blot, respectively. Each bar represents mean ± SE from 3 independent experiments. *P ＜ 0.05, **P ＜ 0.01, compared with control group, ^#P ＜ 0.05, ^##P ＜ 0.01 compared with LPS group, ^лP ＜ 0.05, ^ллP＜0.01 compared with aspirin group.