Abstract:
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.