Fig 2: CI/R activated XBP-1 slicing, neuroinflammation, and neuron pyroptosis in the rat MCAO model. Rats were treated with MCAO. (a) The level of Xbp-1s mRNA in each group with CI/R. (b) The level of NLRP3 mRNA in each group with CI/R. (c) The level of IL-1β mRNA in each group with CI/R. (d) The level of IL-18 mRNA in each group with CI/R. (e) Representative western blots and quantification of the indicated proteins in the hippocampal lysate from rats in each group. Relative expression was normalized to sham rats. (f–k) The expression of XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18 in the hippocampus of the four groups after CI/R injury. Each bar represents the mean ± SEM (n = 6). p > 0.05, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 were statistically significant.

Fig 3: GO and pathway enrichment analysis of genes associated with XBP1. (A) Volcano plot representing genes highly associated with XBP1 using the LinkedOmics database. (B) GO (biological processes) analysis of genes associated with XBP1. GSEA pathway enrichment revealed the genes associated with XBP1 were enriched with response to (C) endoplasmic reticulum stress and (D) protein activation cascade. XBP1s, spliced X-box binding protein 1; GO, Gene Ontology; FDR, false discovery rate.

Fig 4: OGD/R decreased the cell viability and increased cytotoxicity, XBP-1 slicing, and inflammation in HT22 cells. The HT22 cells were, respectively, pretreated with Z-YVAD-FMK (20 μM) and polyphyllin VI (4 μM) 30 min before exposing to OGD/R for 24 h. (a) Cell viability was detected in HT22 cells using a CCK-8 assay. (b) LDH release was detected in HT22 cells using an LDH assay. (c) Representative western blots and quantification of the indicated proteins in the cellular lysate from HT22 cells in each group. Relative expression was normalized to that of the control group. (d–i) The expression of XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18 in HT22 cells after OGD/R. Asterisks indicate statistical significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). All experiments were repeated for three times.

Fig 5: Infection experiments with the neuronal cell line SH-SY5Y were repeated as described in Figure 1, but cells were pretreated with IRE1 inhibitors KIRA6 (KIRA), STF083010 (STF) and GSK2850163 (GSK) for four hours prior to infection as well as after infection or left untreated. Viral RNA was extracted from culture supernatants and cell lysates were collected at 0, 24, 48 and 72 h post infection. Infections of untreated cells with Neudoerfl are shown in green (A), HB171 in blue (B) and LGTV in pink (C). Infections of cells pretreated with KIRA are shown in light brown, STF in light grey and GSK in dark grey, respectively. All infections were performed in triplicates and repeated three times. Statistical analyses were performed by 2-way ANOVA with Tukey’s multiple comparison test, ** p < 0.002, *** p < 0.001. Cell lysates were analyzed by Western blot for XBP1 expression after infection with Neudoerfl (D), HB171 (E) or LGTV (F). GAPDH expression was analyzed as a housekeeping control. A representative blot is shown. kDa, kilodalton.