The 5-HT and PLC Signaling Pathways Regulate the Secretion of IL-1 β, TNF- α and BDNF from NG2 Cells

Abstract

The present study was clarified the relationship between NG2 glial cells and 5-hydroxytryptamine (5-HT) to further revealed a role in the regulation of cortical excitability. The co-localization of NG2 cells and 5-HT in rat prefrontal cortex was determined using immunofluorescence. Different concentrations of 5-HT were applied to cultured NG2 cells. Real-time PCR measured the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and brain-derived neurotrophic factor (BDNF). Changes in the expression of IL-1β, TNF-α, and BDNF in NG2 cells were detected after the addition of 5-HT receptor specific blockers and phospholipase C (PLC) specific activators and inhibitors. The results confirmed that the NG2 protein and 5-HT co-localized in the prefrontal cortex. 5-HT treatment of NG2 cells significantly reduced the expression of IL-1β and BDNF mRNA and increased the expression of TNF-α. The 5-HT receptor specific inhibitors alverine citrate, ketanserin, ondansetron and SB-399885 blocked the regulatory effects of 5-HT on NG2 cells. The PLC signal was linked to the secretion of IL-1β, TNF-α and BDNF in NG2 cells. These results indicated that 5-HT affected IL-1β, TNF-α, and BDNF secretion from NG2 cells via the 5-HT1A, 5-HT2A, 5-HT3, 5-HT6 receptors and the PLC signaling pathway.

Figure 1

Colocalization of NG2 cells and 5-HT neuron markers in the rat prefrontal cortex. Double fluorescence detection of NG2 cells (red; (a)) and the 5-HT neuron marker TPH (green; (b)) in the prefrontal cortex. The merged images show colocalization of NG2 cells and 5-HT neurons in the prefrontal cortex (c). Scale bar = 20 μm.

Figure 2

5-HTRs expressed on NG2 cells. (a) Data presented as expression relative to 5-HT5. (Data are expressed as the means +/− s.d: n = 3 for each group. ^∗p < 0.05, ^∗∗p < 0.01. s.d: standard deviation). (b) The PCR products were visualized in agarose gels. Comparison of the PCR products was performed using GAPDH as a positive control. Controls are labeled as follows: M, DNA marker; 1, 5-HT1AR; 2, 5-HT2AR; 3, 5-HT3R; 4, 5-HT5R; 5, 5-HT6R; 6, 5-HT7R; 7, GAPDH. The product size of each receptor matched the expected product size.

Figure 3

5-HT treatment induces IL-1β, TNF-α and BDNF secretion in NG2 cells. (a-c) IL-1β, TNF-α and BDNF mRNA levels were determined using real-time PCR. (d-f) IL-1β, TNF-α and BDNF protein expression levels were measured using ELISA. (Data are expressed as the means +/− s.d. n = 3 for each group. ^∗p < 0.05, ^∗∗p < 0.01. s.d.: standard deviation).

Figure 4

Effects of various antagonists of 5-HTRs on IL-1β, TNF-α and BDNF secretion from NG2 cells. (a-c) IL-1β, TNF-α and BDNF mRNA levels were estimated using real-time PCR. (d-f) IL-1β, TNF-α and BDNF protein expression was measured using ELISA. (Data are expressed as the means +/− s.d. n = 3 for each group. ^∗p < 0.05, ^∗∗p < 0.01. s.d.: standard deviation).

Figure 5

Effects of the specific activator m-3M3FBS and the specific inhibitor U-73122 of PLC on the secretion of IL-1β, TNF-α and BDNF in NG2 cells. (a-d) Real-time PCR was used to determine the expression of PLC, IL-1β, TNF-α and BDNF mRNA. (d-e) Western blotting was used to determine the expression of PLC, IL-1β, TNF-α and BDNF. GAPDH is shown as an internal control. (Data are expressed as the means +/− s.d. n = 3 for each group. ^∗p < 0.05, ^∗∗p < 0.01. s.d.: standard deviation).