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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: N-Docosahexaenoylethanolamine ameliorates LPS-induced neuroinflammation via cAMP/PKA-dependent signaling

Fig. 3

Involvement of cAMP/PKA signaling in the suppression of LPS-induced TNF-α expression by synaptamide. Microglia cells were incubated with LPS (100 ng/mL) and 10 nM synaptamide, and levels of TNF-α and iNOS mRNA were determined by qPCR at 1 h after incubation. 5-, 12-, 15-LOX inhibitor (2-TEDC, 10 μM); COX inhibitor (Indo, 10 μM); P450 inhibitor (KTZ, 10 μM) (a); adenylyl cyclase inhibitor (SQ 22536, 10 μM); and PKA inhibitor (H-89, 10 μM) (b, c) were added 30 min prior to LPS treatment. BV2 murine microglia cells were transfeted with PKA siRNA or scrambled control RNA for 48 h prior to the treatment with LPS (100 ng/mL) and synaptamide (10 nM) for 1 h (d, e). Successful knockdown of PKA expression is indicated in the inset. PKA primary rat microglia (f, upper) and BV2 (f, bottom) cells were treated with synaptamide (1–100 nM) or forskolin (For, 10 μM) as a positive control for 15 min, and cAMP levels were measured. BV2 cells were incubated with synaptamide at the indicated concentration for 30 min and subjected to immunoblot analysis using antibodies that specifically recognize phosphorylated and non-phosphorylated PKA, CREB (g). Graph values are mean ± SEM of protein levels of p-PKA/p-CREB relative to PKA/CREB, respectively (n = 3). Means designated with the same letter are not significantly different

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