Involvement of oxidative pathways in cytokine-induced secretory phospholipase A2-IIA in astrocytes

Abstract

Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-kappaB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1beta-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1beta-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1beta-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells.

Fig 1

(A) Induction of sPLA2-IIA mRNA in immortalized rat astrocytes (DITNC) by IL-1β and TNFα. Astrocytes were treated with 4 ng/ml with IL-1β or TNFα for 18 h prior to measurement of sPLA2-IIA mRNA expression by RT-PCR. Results are representative of three independent experiments. (B) Induction of sPLA2-IIA protein in astrocytes. DITNC astrocytes were cultured in 60 mm dish and serum-starved for 4 h prior to treatment with TNFα, IL-1β and IFNγ (10 ng/ml) for 48 h. After treatment, culture medium was removed, cells washed with PBS and lysis buffer was added. Medium (40 μl) and cell lysate (15 μg protein) was used in Western blot as described in text. β-actin in cell lysate was used as control.

Fig 2

U0126 (U), a MEK inhibitor, inhibited IL-1β-induced sPLA2-IIA mRNA and protein expressions in astrocytes. (A) For mRNA expression, astrocytes were treated with U0126 (10 μM) alone, IL-1β (4 ng/ml) alone and IL-1β with U0126 (5 and 10 μM) for 18 h. U0126 was added to astrocytes 1 h before treating with IL-1β. sPLA2-IIA expression was measured by real-time PCR relative to β-actin expression as described in text. Results are mean ± SD from three experiments. (B) For protein expression, astrocytes were treated with U0126 (10 μM), IL-1β (10 ng/ml), and IL-1β + U0126 for 48 h. U0126 was added 1 h prior to adding IL-1β. After incubation, cell lysates were taken for Western blot analysis as described in text. β-actin was used as control. Results are mean ± SD from three experiments. *p<0.05, **p<0.01, ***p<0.001 vs. IL-1β.

Fig 3

LY294002, a PI-3 kinase inhibitor, inhibited IL-1β-induced sPLA2-IIA mRNA expression in astrocytes. Astrocytes were treated with LY294002 (LY, 50 μM), IL-1β (4 ng/ml), and IL-1β with LY at 10, 25, and 50 μM for 18 h. LY was added to astrocytes 1 h prior to treatment with IL-1β. sPLA2-IIA mRNA expression was determined by real-time PCR with β-actin as control. Results are mean ± SD from three experiments. ***p<0.001 vs. IL-1β.

Fig 4

Apocynin, an NADPH oxidase inhibitor, inhibited IL-1β-induced sPLA2-IIA mRNA expression in astrocytes. Astrocytes were treated with apocynin (Apo, 1 mM), IL-1β (4 ng/ml), and IL-1β with apocynin at 500, 750 and 1000 μM for 18 h. Apocynin was added to astrocytes 1 h prior to treatment with IL-1β. sPLA2-IIA mRNA expression was determined using real-time PCR with β-actin as control. Results are mean ± SD from three experiments. ***p<0.001 vs. IL-1β.

Fig 5

Resveratrol (Res) inhibited IL-1β-induced sPLA2-IIA mRNA expression in astrocytes. Astrocytes were treated with resveratrol (50 μM), IL-1β (4 ng/ml) and IL-1β with resveratrol at 25 or 50 μM for 18 h. Resveratrol was added to astrocytes 1 h prior to treatment with IL-1β. sPLA2-IIA mRNA expression was determined using real-time PCR with β-actin as control. Results are mean ± SD from three experiments. ***p<0.001 vs. IL-1β.

Fig 6

Epigallocatechin gallate (EGCG) inhibited IL-1β-induced sPLA2-IIA mRNA expression in astrocytes. Astrocytes were treated with EGCG (50 μM), IL-1β (4 ng/ml) and IL-1β with EGCG at 25 or 50 μM for 18 h. EGCG was added to astrocytes 1 h prior to treatment with IL-1β. sPLA2-IIA mRNA expression was determined using real-time PCR with β-actin as control. Results are mean ± SD from three experiments. ***p<0.001 vs. IL-1β.

Fig 7

Resveratrol and EGCG inhibited IL-1β-induced translocation of p67 phox to the membrane fraction. Astrocytes were treated with IL-1β (4 ng/ml) or IL-1β plus Res (50 μM) and EGCG (50 μM) for 10 min. Cells were disrupted and cell cytosol and membrane fractions were separated by centrifugation as described in Method section. (A) Western blot analysis of p67 phox concentration in the membrane fraction compared to β-actin as control. (B) Relative intensity of p67 phox compared to β-actin. Results are mean ± SD from three independent experiments. ***p<0.001 vs. IL-1β.

Fig 8

Apocynin and resveratrol inhibited IL-1β-induced ERK1/2 phosphorylation in astrocytes. Astrocytes were treated with IL-1β (4 ng/ml), or IL-1β plus apocynin (1 mM) and Res (50 μM) for 10 min. (A) Cell lysate was used for Western blot analysis for phospho-ERK1/2 and total ERK1/2. (B) Relative intensities of phospho-p42 ERK against total p42 ERK. Results are mean ± SD from three independent experiments. **p<0.01 vs. IL-1β.