Fig. 6

The anti-inflammatory effect of Fetuin-A is by inhibiting necroptosis in glutamate-exposed microglial cells. The microglial cells were treated with 100 μm glutamate (Glu) for 24 h to induce cellular injury. Meanwhile, Fetuin-A was treated into medium at indicated concentrations. A, B Expression of RIPK3, MLKL, p-RIPK3 and p-MLKL in microglial cells under various treatment conditions. GAPDH was used as the loading control. And bar graphs show the results of analysis (by band density analysis) of these proteins (n = 3). C–F Immunofluorescence assessment of p-RIPK3 and p-MLKL expression. Scale bar is 50 μm. The relative immunofluorescence intensity of Fetuin-A was detected with Image-J software (n = 3). G RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in microglial cells (n = 3). H Transmission electron microscopy (TEM) images of tissues. Translucent cytoplasm, mitochondrial swelling and destruction of membrane integrity were observed in TNF-α + zVAD-treated microglial cells (scale bar = 5 or 1 μm, n = 3). I IL-6, IL-1β, TNF-α, and IL-10 secretion was measured using ELISA (n = 5). Data are presented as the means ± SD; *P < 0.05 vs. control, **P < 0.01 vs. control, ^#P < 0.05 vs. Glu group, and n.s.: no significant difference