Fig. 4

Knockdown of HSPA8 suppresses NF-κB and NLRP3 inflammasome activation in OGD/R-induced primary astrocytes. A Immunofluorescence of GFAP in primary rat spinal cord astrocytes was performed. Scale bar = 100 μm. B The relative protein level of HSPA8 in primary astrocytes was detected after injection of LV-sh-HSPA8 via western blot assay. C The relative protein level of total NF-κB P65, p-NF-κB P65, NLRP3, ASC, and p20/pro-caspase-1 in primary astrocytes was detected after injection of LV-sh-HSPA8 via western blot assay. D The contents of IL-1β and IL-18 in primary astrocytes were detected by ELISA assay. E Relative protein levels of IL-1β and IL-18 in primary astrocytes were detected via western blot assay. F The intracellular location of the NF-κB P65 was observed by immunofluorescence staining. Scale bar = 50 μm. G Quantification of NF-ĸB P65-immunofluorescence intensity in the nuclear was analyzed. H The DNA-binding activity of NF-κB in primary astrocytes was revealed by EMSA. Data were shown as means ± SD of three independent experiments. N = 3 cells per group. Statistical analysis was performed by one-way ANOVA followed by Turkey’s for multiple comparisons. *p < 0.05 vs. sham. ^# p < 0.05 vs. SCII + LV-sh-NC. NLRP3, nod-like receptor pyrin domain-containing 3; ASC, the apoptosis-associated speck-like protein containing CARD; SCII, spinal cord ischemia–reperfusion injury; IL-1β/18, interleukin-1β/18; NF-κB, nuclear factor-kappa B