Roles of reactive oxygen species and protein kinase B phosphorylation in paraquat-mediated microglia activation

Background Paraquat (PQ) can induce microglia activation in central nervous system (CNS). However, its associated mechanism is not clear yet.

Objective This study investigates the potential molecular mechanism of PQ-mediated microglia activation and focuses on the roles of reactive oxygen species (ROS) and protein kinase B (PKB, also called Akt) phosphorylation in this process through in vitro experiments.

Methods Mouse microglia cell line BV-2 cells were treated with different concentrations of PQ (0, 1, 3.3, 10, 33, and 100 μmol·L^-1) for 12 h and assessed for its viability with cell counting kit 8 (CCK8). The highest dose that did not damage the cell viability (33 μmol·L^-1) was selected for further experiments. Then BV-2 cells were treated with 33 μmol·L^-1 PQ for 12 h to detect the levels of nitric oxide synthase (iNOS), a marker of microglia classical activation (M1 activation), CD206, a marker of microglia alternative activation (M2 activation), ROS, and phosphorylated Akt (p-Akt) by flow cytometry, as well as the levels of proinflammatory cytokines triggered via M1 activationinterleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β) and anti-inflammatory cytokines triggered via M2 activationinterleukin 4 (IL-4), insulin-like growth factor 1 (IGF-1), and transforming growth factor β (TGF-β) by ELISA. A batch of cells were pretreated with 2 mmol·L^-1 N-acetylcysteine (NAC) to inhibit ROS for 2 h, and then treated with PQ for 12 h to detect the expression levels of iNOS, CD206, and IL-1β by RT-PCR. Another batch of cells were pretreated with Akt inhibitor (Akt inhibitor Ⅷ, 5 μmol·L^-1) and activator (SC79, 20 μmol·L^-1) respectively for 2 h, and then treated with PQ for another 12 h, to detect the expression levels of iNOS, CD206, and IL-1β.

Results After the cells were exposed to 33 μmol·L^-1 PQ for 12 h, the levels of iNOS (t=8.912, P < 0.001) and proinflammatory cytokines IL-6, TNF-α, and IL-1β were increased (t=2.710, 3.342, and 5.078, P < 0.05), while no significant difference was observed in the levels of CD206 and anti-inflammatory cytokines (P>0.05); in addition, intercellular ROS level was increased (t=11.907, P < 0.001), and Akt phosphorylation was inhibited (t=6.152, P < 0.001). After the NAC treatment, the increased levels of iNOS, IL-1β, and CD206 gene expression induced by PQ were reversed (t=15.457, 6.912, 9.106, respectively; P < 0.001). After the Akt inhibitor treatment, the levels of iNOS and IL-1β were further increased compared with the PQ group (t=8.021, P < 0.001; t=3.684, P < 0.01, respectively), and the CD206 level was reduced (t=2.662, P < 0.05). In contrast, compared with the PQ group, the levels of iNOS and IL-1β were inhibited by the Akt activator (t=6.835, P < 0.001; t=4.325, P < 0.01, respectively), while CD206 was increased (t=17.471, P < 0.001). As for the relationship between ROS and Akt, NAC restored the inhibited Akt phosphorylation induced by PQ (t=6.438, P < 0.001), while neither Akt inhibitor nor Akt activator changed the level of ROS (P < 0.05).

Conclusion PQ can induce pro-inflammatory activation of microglia by increasing ROS and inhibiting Akt phosphorylation. ROS intervention can inhibit both M1 and M2 phenotypes of microglia. And Akt activation can regulate PQ-mediated microglia activation.