HIV-1 Vpr mediated induction of IL-6 and IL-8 in astrocytes involves PI3K/Akt pathway.

SVGA astrocytes were cultured and seeded in 6 well plates. The cells were 1h pre-treated with chemical inhibitor for PI3K/Akt pathway (LY294002) and then transfected with a plasmid encoding HIV-1 Vpr or were mock-transfected. The cells were harvested at 6h post-transfection followed by the determination of IL-6 and IL-8 mRNA expression levels using real-time RT-PCR. The cell culture supernatants were collected at 48 h post-transfection, and protein concentration of secreted IL-6 and IL-8 was determined using BioPlex multi-cytokine assay. (A, C) mRNA expression levels of IL-6 and IL-8 calculated relative to mock-transfected controls in the presence of LY294002, respectively. (B, D) Protein concentrations for secreted IL-6 and IL-8 with LY294002, respectively. (E, F) Depicts the effect of LY294002 on phosphorylated Akt and NF-κB p-65 nuclear translocation, respectively. (G, I) portray mRNA levels while (H, J) show protein concentration for IL-6 and IL-8 when individuals Akt isoforms were silenced, respectively. Every bar represents the mean ± SE of thee independent experiments done in triplicates. Statistical analyses were performed using 1-way ANOVA using post-hoc Tukey HSD test, # p < 0.01, ^a p < 0.05 as compared to Vpr transfected cells; ** p < 0.01 and * p < 0.05 compared to mock-transfected controls.