Figure 1.
sPLA2-IIA inhibition prevents the release of PGE2 in LPS-stimulated brain and astrocytes.
(A) Mice (n = 4) were given i.c.v. injection with LPS (2.5 µg) for 0, 0.5, 1, 4, 24 hours and PGE2 in the cortex were detected by ELISA. Since the levels of PGE2 in the cortex with LPS (2.5 µg) for 1 hour were significantly higher than the control (0 hour), we chose the stimulation of LPS (2.5 µg) for 1 hour in the following experiments in vivo. (B) Inhibition of sPLA2-IIA by SC-215 remarkably reduced the production of PGE2 in the cortex treated with LPS (2.5 µg) for 1 hour. (C) SC-215 lowered LPS-elevated PGE2 levels in a dose dependent manner in primary astrocytes. (D) The sPLA2-IIA protein expression was detected by Western Blot in mice cerebral cortex after treatment with LPS (2.5 µg) for 0, 10, 20, 30, 60 minutes. (E) The LPS-induced sPLA2-IIA upregulation was significantly suppressed by the SC-215 in cerebral cortex. (F) The LPS-induced sPLA2-IIA protein expression was significantly reduced by SC-215 in a dose-dependent manner in primary astrocytes. Values represent the mean ± S.E.M. of results from five animals in each group. One symbol, p<0.05; two symbols, p<0.01; three symbols, p<0.001. Symbols indicate comparison versus either (#) control or (*) LPS.
Figure 2.
Inhibition of sPLA2-IIA by SC-215 reduces LPS-induced phosphorylation of ERK1/2 and cPLA2α.
(A) Astrocytes (n = 3) were incubated for 0 to 4 hours with 100 ng/mL LPS or pretreated with 1.25μM SC-215 for 2 hours before LPS stimulation. (B) Mice (n = 4) were given i.c.v. injection with LPS (2.5 µg) alone for up to 1 hour or injected with SC-215 (1.218 µg) 1 hour before LPS stimulation. (C) In the subsequent experiments, the cortex was obtained 10 mintues or 1 hour later to study the effect of SC-215 on LPS-induced phosphorylated ERK1/2, p38 or cPLA2α respectively. Expression of phosphorylated (p) and total cPLA2α, ERK1/2, and p38 was assessed by Western blotting as described in methods.I.
Figure 3.
ERK1/2 regulates the activation of cPLA2α and the generation of PGE2 in LPS-induced mice and astrocytes.
(A) Mice (n = 4) were given i.c.v. injection with LPS (2.5 µg) alone or injected with AACOCF3 (2 µg) or U0126 (2 µg) 1 hour before LPS stimulation for 1 hour. (B) The in vitro effects of U0126 on PGE2 production and (C) phosphorylation of ERK1/2 and cPLA2α in primary rat astrocytes were determined. Astrocytes (n = 3) were treated with U0126 (01, 1, 2 and 5 µM) for 2 hours before LPS (100 ng/mL) stimulation for up to 24 hours. The PGE2 levels in tissue homogenate at 1 hour after LPS stimulation (A) or culture supernatants at 24 hours after LPS stimulation (B) were determined by using ELISA-based assay as described in methods, the phosphorylation of ERK1/2 and cPLA2α in the cell lysates at 4 hours after LPS challenge (C) were determined by western blot as described in methods. Values represent the mean ± S.E.M. of results from five animals in each group. One symbol, p<0.05; two symbols, p<0.01; three symbols, p<0.001. (#), significant comparisons for LPS versus control ; (*), significant comparisons for U0126 versus LPS; (&), significant comparisons for AACOCF3 versus LPS.
Figure 4.
sPLA2-IIA inhibition prevents LPS-induced expression of COX-2 and mPGES-1.
(A) Mice (n = 4) were given i.c.v. injection with LPS (2.5 µg) alone or injected with SC-215 (2.5 µg)1 hour before LPS stimulation for 1 hour. The cortex was obtained 1 hour later to study the effect of SC-215 on LPS-induced COX-2 and mPGES-1 expression respectively. Protein exptessions were assessed by Western blotting as described in methods.(B) Possible molecular mechanism involved in LPS-induced PGE2 production in astrocytes. LPS activates the sPLA2-IIA, leading to increased levels of phospho-ERK1/2. The accumulation of phosphor-ERK1/2 levels modulates the downstream activates of cPLA2α, cPLA2α regulates COX-2 and mPGES-1 enzyme activations which lead to the production of PGE2. Black solid arrows represent the novelties of the present study. LPS, lipopolysaccharide;, secretory phospholipase A2-IIA; cPLA2α, cytosolic phospholipase A2α; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; mPGES-1, microsomal PGE synthase-1.