Figure 4.

HDAC inhibition indirectly reduces oligodendrocyte injury via microglia after ICH in vitro and modulates microglia/macrophage polarization in primary microglia after ICH through the JAK2/STAT1 pathway. (a) In vitro experiments used a transwell system. Primary microglia were incubated with PBS, scriptaid (1 µM), hemoglobin (20 µM) +PBS and hemoglobin (20 µM) +scriptaid for 6 h and cocultured with primary oligodendrocytes (OLG) in a transwell system. (b) Double immunostaining images of MBP and DAPI in oligodendrocytes that were cocultured with primary microglia stimulated by PBS, scriptaid, hemoglobin +scriptaid and hemoglobin +PBS, respectively (Scale bar = 50 µm). (c) Quantifications of oligodendrocyte survival and cell death with LDH release and CCK8 in the transwell system. (d) RT-PCR demonstrated a decrease in proinflammatory-cytokine genes in the scriptaid-treated group in vitro (d). (e) Double immunostaining of the microglia marker Iba1 and pJAK2 and pSTAT1 in the PBS, Scriptaid, Hemoglobin+PBS and Hemoglobin+Scriptaid groups. (Scale bar=100 µm); quantifications of pSTAT1 and pJAK2 immunoreactivity in primary microglia stimulated by PBS, scriptaid, hemoglobin+scriptaid and hemoglobin+PBS, respectively. (f) Relative protein levels of JAK2, pJAK2, STAT1 and pSTAT1 in primary microglia stimulated by PBS, scriptaid, hemoglobin+scriptaid and hemoglobin+PBS, respectively. n=3–4/group, *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001). All data are presented as mean ± SD.