Fig. 4

Sestrin2 promotes microglial polarization to M2 phenotype to exert neuroprotection after oxygen and glucose deprivation and re-oxygenation. a Diagram of the in vitro experimental design. SESN2 Sestrin2, PBS phosphate buffer saline, CM condition medium. b LDH assay showing death of neurons treated with conditioned medium from OGD-treated BV2 cells supplemented with different doses of sestrin2. c, d Western blot and quantification for cleaved caspase3 in neurons of two groups treated with conditioned medium from BV2 cells culture under OGD conditions or with conditioned medium from OGD-treated cells supplemented with 12.5 ng/ml sestrin2 (OGD + SESN 12.5). C-cas3 cleaved caspase3. e, f Immunostaining images of TUNEL (red), MAP2 (green), and DAPI (blue) in OGD and OGD + SESN 12.5 groups. Scale bar = 50 μm. The bar graph shows the percentage of TUNEL⁺ cells/ DAPI⁺ cells. g The results of the real-time PCR show the mRNA levels of the pro-inflammatory mediators IL-1β, IL-6, and TNF-α and anti-inflammatory mediators IGF-1, IL-10, and TGF-β in BV2 cells treated after OGD with 6.25–100 ng/ml sestrin2 and NS. h Western blot and quantification analysis show the expression of M1-related marker (iNOS) and M1-related marker (Arginase-1) in BV2 cells treated after OGD with 6.25–100 ng/ml sestrin2 and NS. i Immunostaining images of M1 related marker iNOS (red), Iba-1 (green), and DAPI (blue) in control, OGD and OGD + SESN 12.5 groups. Scale bar = 50 μm. Quantification of iNOS⁺ cell number and its percentage to Iba-1⁺ cell number in OGD and OGD+SESN 12.5 groups. j Immunostaining for the M2-related marker CD206 (red), Iba-1 (green) and DAPI (blue) of cells in control, OGD, and OGD + SESN 12.5 groups. Scale bar = 50 μm. Quantification of CD206⁺ cell number and its relative density in OGD and OGD + SESN 12.5 group. N = 4–6 per group. Data are presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001