Fig 2: Change of HSP27 level in rat SAH model. (A) Representative images of rat brain at indicated time from the sham and SAH groups. (B) Samples of CSF was collected (6, 12, 72 h, n = 6; 24 h, n = 7), HSP27 concentration was measured with HSP27 ELISA Kit and expressed in ng/ml. (C,D) Coronal sections from the sham and SAH group reperfusion (12 and 72 h) subjected to immunostaining for the neuronal marker NeuN (green) or macrophages/microglia marker Iba-1 (green) and HSP27 (red) in the basal cortex. Quantification was performed by counting the HSP27/NeuN or HSP27/Iba-1 positive cells per mm2 region in the basal cortex, n = 6, scale bar = 50 µm. Data are mean ± SD, *p < 0.05, ANOVA with Bonferroni's multiple comparisons test.

Fig 3: Effect of HSP27 peptides on hemolysate-induced cell apoptosis in primary cortical neurons. (A) Schematic representation of various HSP27 peptides. (B) Hsp27 peptides have no effect on cell viability in primary cortical neurons; Cortical neurons were treated with indicated HSP27 peptides (0.03 mg/ml) in medium (1:50) for 24 h. Cell viability was measured with Cell Counting Kit-8 (CCK-8) and normalized to control. (C,D) Cortical neurons were treated with hemolysate in medium (1:50) or plus indicated HSP27 peptides (0.03 mg/ml) for 24 h. (C) Active caspase-3 levels in each group were detected by Western blot, ß-actin serves as a control, and quantification of optical density was normalized to control. (D) Representative images of cortical neurons (phase contrast, ×200) and TUNEL staining (red, ×200), and quantification of TUNEL-positive cells from each group was performed. Data are mean ± SD, n = 6, *p < 0.05 vs. hemolysate treatment, ANOVA with Bonferroni's multiple comparisons test.

Fig 4: Effect of HSP27 overexpression on the activation of MKK4, JNK, c-Jun, and caspase-3 on day 2 after SAH. The basal cortex was collected on day 2 following SAH from the sham (n = 6), SAH + vehicle (n = 6), SAH + Con (n = 6), and SAH + HSP27 (n = 7) groups; homogenates were blotted with anti-FLAG, anti-phospho-MKK4, anti-phospho-JNK, anti-phospho-c-Jun, anti-MKK4, anti-active caspase-3, and anti-ß-actin; and quantification of optical density was normalized to sham group. Data are mean ± SD, *p < 0.05, the two-tailed t-test.

Fig 5: AAV-shRNA targeting HSP27 deteriorated neurological deficit after SAH in rats. (A) Representative images of rat brain on day 2 after surgery for the sham, SAH + vehicle, SAH + NC, and SAH + shRNA groups. (B) Behavior scores of each group were assessed at 48 h, n = 6. (C) The basal cortex was collected on day 2 following SAH from the sham (n = 6), SAH + vehicle (n = 6), SAH + NC (n = 6), and SAH + shRNA (n = 5) groups; homogenates were blotted with anti-HSP27, anti-active caspase-3, and anti-ß-actin, quantification of optical density was normalized to sham group. Data are mean ± SD, *p < 0.05, ANOVA with Bonferroni's multiple comparisons test.