PRDX6-iPLA2 aggravates neuroinflammation after ischemic stroke via regulating astrocytes-induced M1 microglia

The crosstalk between astrocytes and microglia is crucial in neuroinflammation following ischemic stroke, with their phenotypes shifting as the disease progresses. One key factor in this process is the activity of peroxiredoxin 6 (PRDX6) phospholipase A2 (iPLA2), which contributes to the generation of reactive oxygen species (ROS). These ROS, in turn, drive the activation of both microglia and astrocytes, although the precise role of PRDX6-iPLA2 in this context has not been fully explored.

To investigate this, we employed MJ33 and a PRDX6 D140A mutation to block PRDX6-iPLA2 activity in vitro and in vivo following ischemic stroke. Additionally, a PRDX6 T177A mutation was used in CTX-TNA2 cell lines to inhibit PRDX6 phosphorylation. Various inhibitors—including NAC, GSK2795039, Mdivi-1, U0126, and SB202190—were applied to block the activities of ROS, NOX2, mitochondrial fission, ERK, and p38, respectively, in CTX-TNA2 cells.

Our results showed that in ischemic stroke, PRDX6 is predominantly expressed in astrocytes, where its iPLA2 activity is involved in activating both astrocytes and microglia. In a co-culture system, the Asp140 mutation in PRDX6 of CTX-TNA2 cells inhibited microglial polarization, reduced ROS production, suppressed NOX2 activation, and prevented Drp1-dependent mitochondrial fission following oxygen-glucose deprivation/reoxygenation (OGD/R). These effects were further enhanced by additional inhibition of ROS production.

Further experiments demonstrated that blocking the phosphorylation of PRDX6 at Thr177 with U0126 and SB202190 reduced PRDX6-iPLA2 activity. Collectively, these findings indicate that PRDX6-iPLA2 plays an important role in astrocyte-induced ROS generation and microglial activation, processes regulated by NOX2 and Drp1-dependent mitochondrial fission pathways. Moreover, the activity of PRDX6-iPLA2 in astrocytes is modulated by MAPK signaling via phosphorylation at Thr177.