Fig. 8.

The effects of P12 on the inflammatory responses in cultured microglia and neurons. (A) The representative TEM images showing the internalization of P12 (red arrows) in the cultured microglia (BV-2) and neurons (HT22) in vitro; cells were treated with P12 (5 nM) for 24 h; the zoom-in image of the black box was shown at the bottom; scale bar = 5 μm (top) and 1 μm (bottom). (B, C) The effects of P12 at different concentrations on the viability of microglial (BV-2) (B) and neuronal (HT22) (C) cells. (D) The effects of P12 on the IL-6 production of BV-2 cells upon LPS (1 μg/mL) stimulation for 4 h. (E, F) Immunoblots showing the effects of P12 on the phosphorylation of p65 (p-p65) and IκBα degradation for NF-κB activation in BV-2 (E) and HT22 (F) cells upon LPS (1 μg/mL) stimulation for different periods (0–2 h); β-actin as the internal control. (G, H) The densitometry analysis of phosphorylated-p65 (G) and IκBα (H) levels in (F); P12 = 5 nM; N = 3 biological replicates. (I) A scheme showing the proposed working mechanisms of P12 in alleviating SAE in septic mice: i) the intraperitoneally injected P12 targeted peritoneal macrophages and regulated their activation to reduce the systemic inflammation and improve the neurobehavioral performance in septic mice; ii) P12 was able to sequester specific proteins (e.g., CCL9 and MMP) in the blood plasma by forming the protein corona, which facilitated P12 internalization by macrophages and reduction of the systemic inflammation; iii) local injection of P12 directly to the brain down-regulated microglia activation and reduced neuroinflammation; the illustration was created with the help of BioRender.com. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.