Fig. 9.

Palmitic acid sensitizes human microglial cells to abnormal responses to cortisol. (A) Representative brightfield images of human microglial cells (HCM3 cells) pretreated with either vehicle (VEH) or 50 μM palmitic acid (PA), followed by treatment with VEH or 100 nM hydrocortisone (CORT) 24 h later. Cells and supernatant were collected 24 h after CORT/VEH treatment. (B) We found an interaction effect on TNF-α mRNA levels (p = 0.0002), indicating that the relationship between PA and TNF-α mRNA expression depends on the presence of CORT. Incubation of PA followed by CORT promoted a dramatic increase in TNF-α mRNA expression relative to control conditions (p = 0.0003). (C) TNF-α protein levels were also significantly influenced by PA (p = 0.0096), CORT (p = 0.0002), and the interaction of PA x CORT (p = 0.0021), partly supporting the mRNA expression results. (D) Reactive Oxygen Species (ROS -intracellular superoxide levels) were detected by dihydroethidium (DHE) measurements, showing increased ROS-positive cells after PA (33% increase) and CORT (19% increase) treatment. PA priming resulted in a 38% increase in ROS + cells after CORT treatment (relative to control conditions). M1 = ROS negative cells; M2 = ROS positive cells. PA increased (E) Quantification of ROS levels normalized by grams of protein demonstrated a significant ROS increase in PA (p < 0.0001) and CORT (p < 0.0001) treated cells. We also observed a significant interaction effect (p = 0.0459). RFU: relative fluorescence unit. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001. Sample size = four independent experiments. Error bars represent standard errors.